Bone lesions and intestinal barrier disruption caused by the isolated novel goose parvovirus infection in ducks.

Short beak and dwarfism syndrome (SBDS) is attributed to Novel Goose Parvovirus (NGPV), which has inflicted significant economic losses on farming in China. Despite its significant impact, limited research has been conducted on the pathogenesis of this disease. The SD strain, a parvovirus variant isolated from ducks in Shandong province, was identified and characterized in our study. Phylogenetic analysis and sequence comparisons confirmed the classification of the SD strain as a member of NGPV. Based on this information, we established an animal model of SBDS by inoculating Cherry Valley ducks with the SD strain. Our findings indicate that infection with the SD strain leads to a reduction in body weight, beak length, width, and tibia length. Notably, significant histopathological alterations were observed in the thymus, spleen, and intestine of the infected ducks. Furthermore, the SD strain induces bone disorders and inflammatory responses. To evaluate the impact of NGPV on intestinal homeostasis, we performed 16S rDNA sequencing and gas chromatography to analyze the composition of intestinal flora and levels of short-chain fatty acids (SCFAs) in the cecal contents. Our findings revealed that SD strain infection induces dysbiosis in cecal microbial and a decrease in SCFAs production. Subsequent analysis revealed a significant correlation between bacterial genera and the clinical symptoms in NGPV SD infected ducks. Our research providing novel insights into clinical pathology of NGPV in ducks and providing a foundation for the research of NGPV treatment targeting gut microbiota.

Effect of persistent malnutrition on pulmonary tuberculosis treatment: A cross-sectional study in Weifang, China.

BACKGROUND AND OBJECTIVES: Malnutrition is associated with pulmonary tuberculosis (PTB). The aim of this study is to investigate the association between persistent malnutrition and the effect of PTB treatment. METHODS AND STUDY DESIGN: A total of 915 PTB patients were included. Baseline demographic information, anthropometry, and nutritional indicators were measured. The treatment effect was assessed by combinations of clinical manifestations, sputum smear, chest computerized tomography, gastrointestinal symptoms, and the indexes of liver function. Persistent malnutrition was considered when one or more indicators of malnutrition were lower than the reference standards in two tests on admission and after one month of treatment. Clinical symptom score (TB score) was used to assess the clinical manifestations. The generalized estimating equation (GEE) was used to assess the associations. RESULTS: In GEE analyses, patients with underweight had a higher incidence of TB score >3 (OR=2.95; 95% CI, 2.28-3.82) and lung cavitation (OR=1.36; 95% CI, 1.05-1.76). Hypoproteinemia was associated with a higher risk of TB score >3 (OR=2.73; 95% CI, 2.08-3.59) and sputum positive (OR=2.69; 95% CI, 2.08-3.49). Anemia was associated with a higher risk of TB score >3 (OR=1.73; 95% CI, 1.33-2.26), lung cavitation (OR=1.39; 95% CI, 1.19-1.63), and sputum positive (OR=2.23; 95% CI, 1.72-2.88). Lymphocytopenia was associated with a higher risk of gastrointestinal adverse reactions (OR=1.47; 95% CI, 1.17-1.83). CONCLUSIONS: Persistent malnutrition within one month of treatment can adversely affect anti-tuberculosis treatment. Nutritional status during anti-tuberculosis treatment should be continuously monitored.

Improved Dual Attention for Anchor-Free Object Detection.

In anchor-free object detection, the center regions of bounding boxes are often highly weighted to enhance detection quality. However, the central area may become less significant in some situations. In this paper, we propose a novel dual attention-based approach for the adaptive weight assignment within bounding boxes. The proposed improved dual attention mechanism allows us to thoroughly untie spatial and channel attention and resolve the confusion issue, thus it becomes easier to obtain the proper attention weights. Specifically, we build an end-to-end network consisting of backbone, feature pyramid, adaptive weight assignment based on dual attention, regression, and classification. In the adaptive weight assignment module based on dual attention, a parallel framework with the depthwise convolution for spatial attention and the 1D convolution for channel attention is applied. The depthwise convolution, instead of standard convolution, helps prevent the interference between spatial and channel attention. The 1D convolution, instead of fully connected layer, is experimentally proved to be both efficient and effective. With the adaptive and proper attention, the correctness of object detection can be further improved. On public MS-COCO dataset, our approach obtains an average precision of 52.7%, achieving a great increment compared with other anchor-free object detectors.

Bioinspired Total Synthesis of (+)-Euphorikanin A.

We have achieved a bioinspired total synthesis of (+)-euphorikanin A, which possesses an intriguing and complex 5/6/7/3-fused tetracyclic skeleton bearing a bridged [3.2.1]-γ-lactone moiety. Key transformations include stereoselective alkylation and aldol condensation to install the main stereocenters, an intramolecular nucleophile-catalyzed aldol lactonization of carboxylic acid-ketone to assemble the five-membered ring, a McMurry coupling to construct the seven-membered ring, and a biomimetic benzilic acid type rearrangement to form the bridged [3.2.1]-γ-lactone moiety.

RatioNet: Ratio Prediction Network for Object Detection.

In object detection of remote sensing images, anchor-free detectors often suffer from false boxes and sample imbalance, due to the use of single oriented features and the key point-based boxing strategy. This paper presents a simple and effective anchor-free approach-RatioNet with less parameters and higher accuracy for sensing images, which assigns all points in ground-truth boxes as positive samples to alleviate the problem of sample imbalance. In dealing with false boxes from single oriented features, global features of objects is investigated to build a novel regression to predict boxes by predicting width and height of objects and corresponding ratios of l_ratio and t_ratio, which reflect the location of objects. Besides, we introduce ratio-center to assign different weights to pixels, which successfully preserves high-quality boxes and effectively facilitates the performance. On the MS-COCO test-dev set, the proposed RatioNet achieves 49.7% AP.

Complete genome and molecular characterization of genotype VII velogenic Newcastle disease virus isolated in China.

Circulation of dominant genotype VII of Newcastle disease virus (NDV) causes significant economic losses to the poultry industry in China. Although most of genotype VII NDV has frequently been isolated in China to date, the genome sequence difference between duck-origin and chicken-origin NDVs remains largely unknown. In this study, a NDV strain of Chicken/China/HB/2017 (HB), isolated during an outbreak in China, was subjected to genetic, biological, phylogenetic and the pathogenicity characterization. The complete genome of HB strain is 15,192 nucleotides (nt) long and consisting of six genes in the order of 3'-NP-P-M-F-HN-L-5'. Several amino acid mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains, and neutralizing epitopes. Phylogenetic analysis based on the F gene revealed that the HB strain and three other duck-origin NDV strains in China were grouped under subgenotype VII.1.1 and shared 99.1~99.2% nucleotide identity. Additionally, the challenge experiment results showed that the strain was highly pathogenic with 100% morbidity and mortality. Virus shedding was detected from 2 days post-infection until the fifth day. In conclusion, this study offers our understanding of circulating strains of NDV and genes involved in virulence and evolution between different hosts. Keywords: Newcastle disease virus; China; complete genome; genotype VII; mutations.

Immunosuppressive Nor-isopimarane Diterpenes from Cultures of the Fungicolous Fungus Xylaria longipes HFG1018.

Eighteen new nor-isopimarane diterpenes, xylarinorditerpenes A-R (1-18), along with two previously reported compounds, 14α,16-epoxy-18-norisopimar-7-en-4α-ol (19) and the labdane-type diterpene agatadiol (20), were isolated from cultures of the fungicolous fungus Xylaria longipes HFG1018 isolated from the wood-rotting basidiomycete Fomitopsis betulinus. The structure elucidation and relative configuration assignments of 1-18 were accomplished by interpretation of spectroscopic data and through computational methods. The absolute configurations of 1, 4, and 16 were determined by single-crystal X-ray diffraction. Compounds 1-16 possess an 18- or 19-nor-isopimarane skeleton, and compounds 17 and 18 possess an 18,19-dinor-isopimarane skeleton. Compounds 2-5, 9, 14, 19, and 20 showed immunosuppressive activity but were devoid of cytotoxicity against the cell proliferation by concanavalin A-induced T lymphocytes and lipopolysaccharide-induced B lymphocytes, with IC50 values varying from 1.0 to 27.2 µM and from 16.1 to 51.8 µM, respectively.

Two Residues in NSP9 Contribute to the Enhanced Replication and Pathogenicity of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) possesses greater replicative capacity and pathogenicity than classical PRRSV. However, the factors that lead to enhanced replication and pathogenicity remain unclear. In our study, an alignment of all available full-length sequences of North American-type PRRSVs (n = 204) revealed two consistent amino acid mutations that differed between HP-PRRSV and classical PRRSV and were located at positions 519 and 544 in nonstructural protein 9. Next, a series of mutant viruses with either single or double amino acid replacements were generated from HP-PRRSV HuN4 and classical PRRSV CH-1a infectious cDNA clones. Deletion of either of the amino acids led to a complete loss of virus viability. In both Marc-145 and porcine alveolar macrophages, the replicative efficiencies of mutant viruses based on HuN4 were reduced compared to the parent, whereas the replication level of CH-1a-derived mutant viruses was increased. Plaque growth assays showed clear differences between mutant and parental viruses. In infected piglets, the pathogenicity of HuN4-derived mutant viruses, assessed through clinical symptoms, viral load in sera, histopathology examination, and thymus atrophy, was reduced. Our results indicate that the amino acids at positions 519 and 544 in NSP9 are involved in the replication efficiency of HP-PRRSV and contribute to enhanced pathogenicity. This study is the first to identify specific amino acids involved in PRRSV replication or pathogenicity. These findings will contribute to understanding the molecular mechanisms of PRRSV replication and pathogenicity, leading to better therapeutic and prognostic options to combat the virus.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a significant threat to the global pig industry. Highly pathogenic PRRSV (HP-PRRSV) first emerged in China in 2006 and has subsequently spread across Asia, causing considerable damage to local economies. HP-PRRSV strains possess a greater replication capacity and higher pathogenicity than classical PRRSV strains, although the mechanisms that underlie these characteristics are unclear. In the present study, we identified two mutations in HP-PRRSV strains that distinguish them from classical PRRSV strains. Further experiments that swapped the two mutations in an HP-PRRSV strain and a classical PRRSV strain demonstrated that they are involved in the replication efficiency of the virus and its virulence. Our findings have important implications for understanding the molecular mechanisms of PRRSV replication and pathogenicity and also provide new avenues of research for the study of other viruses.

CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

Several groups have used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for DNA virus editing. In most cases, one single-guide RNA (sgRNA) is used, which produces inconsistencies in gene editing. In this study, we used a swine herpesvirus, pseudorabies virus, as a model to systematically explore the application of CRISPR/Cas9 in DNA virus editing. In our current report, we demonstrated that cotransfection of 2 sgRNAs and a viral genome resulted in significantly better knockout efficiency than the transfection-infection-based approach. This method could result in 100% knockout of ≤3500 bp of viral nonessential large fragments. Furthermore, knockin efficiency was significantly improved by using 2 sgRNAs and was also correlated with the number of background viruses. We also demonstrated that the background viruses were all 2-sgRNA-mediated knockout mutants. Finally, this study demonstrated that the efficacy of gene knockin is determined by the replicative kinetics of background viruses. We propose that CRISPR/Cas9 coupled with 2 sgRNAs creates a powerful tool for DNA virus editing and offers great potential for future applications.-Tang, Y.-D., Guo, J.-C., Wang, T.-Y., Zhao, K., Liu, J.-T., Gao, J.-C., Tian, Z.-J., An, T.-Q., Cai, X.-H. CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

MOV10 inhibits replication of porcine reproductive and respiratory syndrome virus by retaining viral nucleocapsid protein in the cytoplasm of Marc-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been a major threat to global industrial pig farming ever since its emergence in the late 1980s. Identification of sustainable and effective control measures against PRRSV transmission is a pressing problem. The nucleocapsid (N) protein of PRRSV is specifically localized in the cytoplasm and nucleus of virus-infected cells which is important for PRRSV replication. In the current study, a new host restricted factor, Moloney leukemia virus 10-like protein (MOV10), was identified as an inhibitor of PRRSV replication. N protein levels and viral replication were significantly reduced in Marc-145 cells stably overexpressing MOV10 compared with those in wild-type Marc-145 cells. Adsorption experiments revealed that MOV10 did not affect the attachment and internalization of PRRSV. Co-immunoprecipitation and immunofluorescence co-localization analyses showed that MOV10 interacted and co-localized with the PRRSV N protein in the cytoplasm. Notably, MOV10 affected the distribution of N protein in the cytoplasm and nucleus, leading to the retention of N protein in the former. Taken together, these findings demonstrate for the first time that MOV10 inhibits PRRSV replication by restricting the nuclear import of N protein. These observations have great implications for the development of anti-PRRSV drugs and provide new insight into the role of N protein in PRRSV biology.

Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important globally distributed and highly contagious pathogen that has restricted cell tropism in vivo and in vitro. In the present study, we found that annexin A2 (ANXA2) is upregulated expressed in porcine alveolar macrophages infected with PRRSV. Additionally, PRRSV replication was significantly suppressed after reducing ANXA2 expression in Marc-145 cells using siRNA. Bioinformatics analysis indicated that ANXA2 may be relevant to vimentin, a cellular cytoskeleton component that is thought to be involved in the infectivity and replication of PRRSV. Co-immunoprecipitation assays and confocal analysis confirmed that ANXA2 interacts with vimentin, with further experiments indicating that the B domain (109-174 aa) of ANXA2 contributes to this interaction. Importantly, neither ANXA2 nor vimentin alone could bind to PRRSV and only in the presence of ANXA2 could vimentin interact with the N protein of PRRSV. No binding to the GP2, GP3, GP5, nor M proteins of PRRSV was observed. In conclusion, ANXA2 can interact with vimentin and enhance PRRSV growth. This contributes to the regulation of PRRSV replication in infected cells and may have implications for the future antiviral strategies.

Correction to: Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication.

In the original publication of this article [1], the author found the brand of vimentin antibody was wrong in Fig. 3. The legend of Fig. 3, 'mouse anti-vimentin mAb (Cell Signaling Technology) at 4 °C overnight' should be 'mouse anti-vimentin mAb (Sigma-Aldrich) at 4 °C overnight'.

Ecological and physical barriers shape genetic structure of the Alpine porcini (Boletus reticuloceps).

The Alpine porcini, Boletus reticuloceps, is an ectomycorrhizal mushroom distributed in subalpine areas of Southwest China, central China, and Taiwan Island. This distribution pattern makes it an ideal organism to infer how ectomycorrhizal fungi have reacted to historical tectonic and climatic changes, and to illustrate the mechanism for the disjunction of organisms between Southwest China and Taiwan. In this study, we explored the phylogeographic pattern of B. reticuloceps by microsatellite genotyping, DNA sequencing, ecological factor analysis, and species distribution modeling. Three genetic groups from the East Himalayas (EH), northern Hengduan Mountains (NHM), and southern Hengduan Mountains (SHM), were identified. The earlier divergent SHM group is found under Abies in moister environments, whereas the EH and NHM groups, which are physically separated by the Mekong-Salween Divide, are found mainly under Picea in drier environments. Samples from Taiwan showed a close relationship with the SHM group. High mountains did not form dispersal barriers among populations in each of the EH, NHM, and SHM groups, probably due to the relatively weak host specificity of B. reticuloceps. Our study indicated that ecological heterogeneity could have contributed to the divergence between the SHM and the NHM-EH groups, while physical barriers could have led to the divergence of the NHM and the EH groups. Dispersal into Taiwan via Central China during the Quaternary glaciations is likely to have shaped its disjunct distribution.

Importation and Recombination Are Responsible for the Latest Emergence of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus in China.

In China, a majority of the highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRSV) strains were seeded by the 2006 outbreak. However, the most recently emerged (2013-2014) HP-PRRSV strain has a very different genetic background. It is a NADC30-like PRRSV strain recently introduced from North America that has undergone genetic exchange with the classic HP-PRRSV strains in China. Subsequent isolation and characterization of this variant suggest high pathogenicity, so it merits special attention in control and vaccine strategies.

Complete mitochondrial genome sequence and phylogenetic analysis of Tylopilus brunneirubens (Boletales, Basidiomycota).

Tylopilus brunneirubens is a common species in southern China. It is known for brown to dark brown pileus, white context turning reddish brown or rust brown when touched and distinct reticulation on the upper stem. However, little is known about its mitochondrial genome and its relationship with other boletes. Our analysis revealed that the mitochondrial genome of this species is a circular DNA molecule that spans 32,389 bp. It contains 15 core protein-coding genes, 24 transfer RNA genes, and two ribosomal RNA genes. The base composition of the mitochondrial genome is as follows: A (37.20%), C (11.32%), G (12.48%), and T (39.00%), with a GC content of 23.80%. Furthermore, a phylogenetic tree based on 24 mitochondrial genomes provided valuable insights into the phylogenetic relationships of Tylopilus brunneirubens with other boletes for the first time.

Complete mitochondrial genome sequence of Butyriboletus hainanensis (Boletales, Basidiomycota).

Butyriboletus hainanensis, a macrofungus belonging to the Boletaceae family, is named after its collection location on Hainan Island, China. However, little is known about its mitochondrial genome and its phylogenetic relationship with other boletes. In this study, we utilized next-generation sequencing technology to sequence the mitochondrial genome of Bu. hainanensis. Our findings revealed that the mitochondrial genome of this species is presumably a circular DNA molecule spanning 36,592 bp. It consists of 15 protein-coding genes, 27 transfer RNA genes, and two ribosomal RNA genes. The base composition of the mitochondrial genome is as follows: A (36.64%), C (12.22%), G (11.73%), and T (39.41%), with a GC content of 23.95%. Additionally, a phylogenetic tree was constructed based on 22 mitochondrial genomes, which provided valuable insights into the phylogenetic relationships of Bu. hainanensis with other boletes for the first time.

Complete mitochondrial genome sequence of Aureoboletus raphanaceus (Boletales, Basidiomycota).

Aureoboletus raphanaceus is a member of boletoid mushroom, which is named after its distinctive radish smell. The mitochondrial genome and phylogenetic relationships with other boletes need to be investigated to gain a comprehensive understanding of it. In this study, we sequenced the mitochondrial genome of A. raphanaceus using next-generation sequencing technology and found that its mitochondrial genome is a circular DNA molecule measuring 42,157 bp. It consists of 15 core protein-coding genes, 27 transfer RNA genes, and two ribosomal RNA genes. The mitochondrial genome had a base composition of A (39.89%), C (11.06%), G (11.67%), and T (37.38%), with a GC content of 22.73%. A phylogenetic tree based on 22 mitochondrial genomes was constructed, which provided the first insights into the phylogenetic relationships of this species with related boletes.

A novel subunit vaccine based on Fiber1/2 knob domain provides full protection against fowl adenovirus serotype 4 and induces stronger immune responses than a Fiber2 subunit vaccine.

Outbreaks of hepatitis-hydropericardium syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry in China since 2015. However, commercially available vaccines against the FAdV-4 infection remain scarce. In our study, subunit vaccine candidates derived from the bacterially expressed recombinant Fiber1 knob domain and Fiber2 knob domain fusion protein (termed as Fiber1/2 knob subunit vaccine) and Fiber2 protein (termed as Fiber2 subunit vaccine) of the FAdV-4 SDSX strain were developed. Immunogenicity evaluation showed that the Fiber1/2 knob subunit vaccine induced the production of antibodies at 7 d postvaccination (dpv), earlier than the Fiber2 subunit vaccine. Moreover, the neutralizing antibody level of the Fiber1/2 subunit vaccine group was higher than the Fiber2 subunit vaccine group, showing significant differences at 14, 21, and 28 dpv. Immune protection test results revealed that both Fiber1/2 knob subunit and Fiber2 subunit vaccines could protect chickens from death against FAdV-4 challenge, although the weight of chickens in the Fiber1/2 knob subunit vaccine group decreased less. Furthermore, analysis of plasma Glutamic oxaloacetic transaminase (AST) and blood glutamic pyruvic transaminase (ALT) levels suggested that the Fiber1/2 subunit vaccine can significantly inhibit liver damage caused by FAdV-4 infection and is more effective in blocking the pathogenicity of FAdV-4 in target organs. In addition, the Fiber1/2 knob subunit vaccine further reduced the viral load in different tissues and virus shedding in chickens than the Fiber2 subunit vaccine. Overall, the Fiber1/2 knob subunit vaccine was more effective than the Fiber2 subunit vaccine. These findings lay the foundation for the development of more effective FAdV-4 subunit vaccines.

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope 110DLDGAW115 was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.

Poria cocos polysaccharide-loaded Alum Pickering emulsion as vaccine adjuvant to enhance immune responses.

Traditional Alum adjuvants mainly elicit a Th2 humoral immune response, but fail to generate a robust Th1 cellular immune response. However, the cellular immune response is essential for vaccination against cancer and a number of chronic infectious diseases, including human immunodeficiency virus infection and tuberculosis. In our previous study, we demonstrated that the polysaccharide from Poria cocos (PCP) has the potential to serve as an immunologic stimulant, enhancing both humoral and cellular immune responses. However, this effect was only observed at high concentrations. In this study, to enhance the immune-stimulation effect of PCP and modify the type of immune response elicited by Alum adjuvant, we successfully developed a Pickering emulsion delivery system (PCP-Al-Pickering) using PCP-loaded Alhydrogel particles as the stabilizer. After optimization, the Pickering emulsion exhibited excellent storage capacity and effectively adsorbed the PCP and antigen. As an adjuvant delivery system, the PCP-Al-Pickering emulsion facilitated the antigen uptake by macrophages, increased the recruitment of cells at injection sites, improved the activation of dendritic cells in draining lymph nodes, elicited a potent and durable antibody response, and promoted the activation of CD4+ and CD8+ T cells. Importantly, the PCP-Al-Pickering emulsion adjuvant elicited a balanced Th1 and Th2 immune response, in comparison to Alum adjuvant. The PCP-Al-Pickering emulsion may serve as a safe and promising adjuvant delivery system to enhance immune responses.