Abstract Complete ammonia oxidizers (comammox) are widespread nitrifiers that can dominate ammonia oxidation in diverse environments by efficiently converting ammonia to nitrate within a single cell, yet the molecular basis distinguishing their ammonia monooxygenase (AMO) from those of canonical bacterial AMO remains unresolved. Here we report cryo-electron microscopy (cryo-EM) structures of AMO from the comammox Nitrospira inopinata ( Ni AMO) in inhibitor-free and allylthiourea (ATU)-bound states at 2.47 Å and 2.68 Å resolution, respectively. Ni AMO displays distinctive auxiliary-subunit organization, copper-site configuration and hydrophobic-channel architecture. Integrative molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations support a methyl-plastoquinol (methyl-PQH 2 )-coupled, Cu D -centric catalytic model, with Cu C potentially facilitating quinone redox cycling. N. inopinata exhibited broad susceptibility to several known nitrification inhibitors, and ATU-bound Ni AMO structure localized the inhibitor to the Cu C –Cu D region, accompanied by constriction of the hydrophobic channel, which is consistent with the competitive role of ATU demonstrated in recovery assays. Multi-omics analyses further revealed an energy-limited stress response to ATU, including induction of urea transport and utilization systems. Collectively, these findings define a methyl-PQ-linked catalytic and inhibitor-responsive architecture of comammox AMO and establish a mechanistic framework for lineage-aware management of nitrification in natural and engineered ecosystems.

PldB, a type VI secretion system-dependent phospholipase D (PLD) effector secreted by multidrug-resistant Pseudomonas aeruginosa, alters host cell membrane permeability and facilitates pathogen internalization. Its cytotoxic activity is neutralized by three cognate immunity proteins-PA5086, PA5087, and PA5088-which protect the bacterium from self-intoxication. However, the underlying mechanism remains unclear. Through quantitative and qualitative analyses, we demonstrate that these three immunity proteins function cooperatively to inhibit PldB toxicity. Cryogenic electron microscopy of the PldB-PA5088 complex reveals that PA5088 binds to the HKD2 domain of PldB primarily through electrostatic interactions, markedly reducing the volume of its active center. Interaction studies using domain‑specific truncated PldB variants, together with enzyme activity assays, identify distinct copy numbers and binding regions for PA5086, PA5087, and PA5088 in their association with PldB. Collectively, our findings provide mechanistic insights into immunity protein-mediated neutralization of PldB toxicity, offering a potential foundation for designing PLD-targeting therapeutics against P. aeruginosa infection.

Summary Ammonia oxidation initiates nitrification and is closely linked to microbial N 2 O production. Ammonia monooxygenase (AMO) catalyzes the first and rate-limiting step of nitrification and is widespread across evolutionarily distinct ammonia-oxidizing archaea (AOA) and bacteria (AOB). The ocean is the largest biome for AOA and AOB, which have distinct ecological niches and markedly different sensitivities to nitrification inhibitors. However, the lack of archaeal AMO structures and inhibitor-bound AMO complexes has hindered mechanistic understanding of the architectural, catalytic, and inhibitory divergence between these two enzyme systems. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of marine archaeal AMO captured in active and inactivated states within its native membrane environment, together with inhibitor-bound structures of estuarine bacterial AMO. Archaeal AMO forms an unexpected cup-shaped homotrimer composed of eight subunits per protomer and exhibits substantial architectural divergence from bacterial AMO. Integrated structural, biochemical, kinetic, and computational analyses reveal distinct periplasmic architectures, copper-center organization, and hydrophobic channels between archaeal and bacterial AMOs for ammonium acquisition, catalysis and inhibitor response. These findings provide a structural and mechanistic framework for understanding how archaeal and bacterial AMOs have diverged to distinct ammonia-oxidizing strategies and inhibitor susceptibilities across environmentally important ammonia oxidizers.

Ammonia monooxygenase (AMO) oxidizes ammonia to hydroxylamine. Limited knowledge of the structural information of AMO hinders our understanding of the molecular mechanism underlying ammonia oxidation, impacting the mitigation of greenhouse gas emissions and enhancing agricultural productivity using ammonium as a nitrogen source. Herein, we report the cryo-electron microscopy structure of the AMO complex from an isolated strain of ammonia-oxidizing bacteria (AOB). AMO is a cylinder-shaped homotrimeric assembly composed of five subunits. A single-transmembrane protein and a soluble protein are potentially crucial in signal transduction during ammonia oxidation and mediating interactions with the outer membrane protein assembly machinery. Three modeled coppers, along with an adjacent water-mediated hydrogen-bond network, may facilitate an efficient proton transfer pathway from the periplasmic CuB to the active site CuD within the inner membrane, where CuC and CuD will act in concert to catalyze substrate reaction. The distinctive surface charge characteristics of AMO provide valuable insights into the structural features that govern ammonium assimilation and material transport during ammonia oxidation. These findings shed light on the molecular complexities of AMO and provides a structural foundation for elucidating the catalytic mechanism of ammonia oxidation.

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), and classical swine fever virus (CSFV) are currently prevalent worldwide and cause similar neurological symptoms in infected pigs. It is very important to establish a detection method that can rapidly and accurately detect and differentiate these three viruses. Targeting the PHEV N gene, PRV gB gene, and CSFV 5 ' untranslated region (5 ' UTR), three pairs of specific primers and probes were designed, and a triplex crystal digital reverse transcription-PCR (cdRT-PCR) was developed to detect PHEV, PRV, and CSFV. The results indicated that this assay had high sensitivity, and the limitation of detection (LODs) for PHEV, PRV, and CSFV were 4.812, 4.047, and 5.243 copies/reaction, respectively, which was about 50 times higher than that of multiplex real-time quantitative RT-PCR (RT-qPCR). This assay showed good specificity, without cross-reaction with other important swine pathogens, i.e., FMDV, PRRSV, PEDV, SIV, TGEV, PoRV, and PCV2. This assay had high repeatability, with intra-assay coefficients of variation (CVs) of 0.73-1.87%, and inter-assay CVs of 0.57-2.95%. The developed assay was used to test 1,367 clinical tissue samples from Guangxi province in China, and the positive rates of PHEV, PRV, and CSFV were 3.44% (47/1,367), 1.24% (17/1,367), and 1.90% (26/1,367), respectively, with a coincidence rate of 98.98% and a Kappa value of 0.94 to the reference multiplex RT-qPCR. The established triplex cdRT-PCR was a highly rapid, sensitive, and accurate assay to detect and differentiate PHEV, PRV, and CSFV.

Canine coronavirus (CCoV) is an important gastrointestinal pathogen that causes serious harm to pet dogs worldwide. Here, 1791 clinical samples were collected from pet dogs in different pet hospitals in Guangxi Province, southern China, for the 2021–2024 period and detected for CCoV by a multiplex RT-qPCR. The results showed that 8.43% (151/1791) of samples were positive for CCoV. Sixty-five positive samples were selected to amplify, sequence, and analyze S, M, and N genes. A sequence comparison revealed that the nucleotide and amino acid similarities of the S, M, and N genes were 94.86% and 94.62%, 96.85% and 97.80%, and 96.85% and 97.80%, respectively. Phylogenetic analysis indicated that 65 CCoV strains obtained in this study belonged to the CCoV-II genotype, of which 56 CCoV strains belonged to the CCoV-IIa subtype and 9 CCoV strains belonged to the CCoV-IIb subtype. A potential recombination event analysis of S gene sequences indicated that two CCoV strains, i.e., GXBSHM0328-34 and GXYLAC0318-35, have recombination signals. A Bayesian analysis indicated that the evolutionary rates of the S, M, and N genes were 1.791 × 10−3, 6.529 × 10−4, and 4.775 × 10−4 substitutions/site/year, respectively. The population size grew slowly before 1980 and then began to shrink slowly; it then shrank rapidly in 2005 and expanded sharply in 2020, leveling off thereafter. These results indicated the CCoV strains prevalent in Guangxi Province, southern China, showed a high level of genetic diversity and maintained continuous variation among clinical epidemic strains.

Background and Aim: Feline coronavirus (FCoV), feline panleukopenia virus (FPV), and feline leukemia virus (FeLV) are prevalent throughout China and significantly threaten cat health. These viruses cause similar manifestations and pathological damage. Rapid and accurate diagnosis depends on detection in the laboratory. This study aimed to establish a reliable and rapid method for accurate detection of FCoV, FPV, and FeLV so that a definite diagnosis can be made and effective measures can be taken to prevent and control viral infection. Materials and Methods: We designed three pairs of specific primers and probes for the detection of FCoV 5 ' untranslated region, FPV viral protein 2, and FeLV pol genes. Recombinant plasmid constructs were generated for use as standard plasmid constructs. Optimal reaction conditions, including primer and probe concentrations, reaction cycles, and annealing temperatures, were obtained on the basis of optimization tests. One-step triplex real-time reverse transcription -quantitative polymerase chain reaction (RT-qPCR) was successfully established to simultaneously detect FCoV, FPV, and FeLV. The specificity, sensitivity, and repeatability of the assay were analyzed, and its applicability was validated by testing 1175 clinical samples. Results: One-step triplex RT-qPCR had a high degree of specificity only for the detection of FCoV, FPV, and FeLV; it had high sensitivity with limits of detection of 139.904, 143.099, and 152.079 copies/reaction for p-FCoV, p-FPV, and p-FeLV standard plasmid constructs, respectively, and it had reliable repeatability with 0.06%-0.87% intra-assay coefficients of variations. A total of 1175 clinical samples were examined for FCoV, FPV, and FeLV using triplex RT-qPCR, and the FCoV, FPV, and FeLV positivity rates were 18.47%, 19.91%, and 47.57%, respectively. The clinical sensitivity and specificity of one-step triplex RT-qPCR were 93.07% and 97.99%, respectively. Conclusion: We developed a rapid and reliable one-step triplex RT-qPCR method for the detection of FCoV, FPV, and FeLV, which could be used as a diagnostic tool for clinical monitoring and diagnosis.

Background: Canine coronavirus (CCoV), canine rotavirus (CRV), canine parvovirus (CPV), and canine distemper virus (CDV) cause gastroenteritis in dogs, and co-infections of these pathogens are common in China. In particular, CCoV and CRV are confirmed to have important zoonotic potential and cause public health issues. It is difficult to diagnose these diseases based only on clinical manifestations and pathological damage. Methods: In this study, four pairs of specific primers and probes targeting the CCoV M, CRV VP7, CPV VP2, and CDV N genes were designed. The reaction conditions, including the primer and probe concentrations, annealing temperatures, and reaction cycles, were optimized for the development of a quadruplex RT-qPCR for the detection of CCoV, CRV, CPV, and CDV. The assay was used to test 1028 clinical samples to validate its application. Results: A quadruplex RT-qPCR was successfully established for the differential detection of CCoV, CRV, CPV, and CDV, with good specificity, high sensitivity, and excellent repeatability. The assay could specifically detect CCoV, CRV, CPV, and CDV without cross-reactivity with the other canine viruses tested. It showed high sensitivity with limits of detection (LOD) of 1.1 × 102 copies/reaction for all four plasmid constructs. It showed excellent repeatability, with 0.05–0.90% intra-assay variation and 0.02–0.94% inter-assay variation. The 1028 clinical samples were tested using the quadruplex RT-qPCR and a reported reference RT-qPCR. The positivity rates of CCoV, CRV, CPV, and CDV were 9.53%, 0.97%, 25.68%, and 5.06% using the developed assay, and 9.05%, 0.88%, 25.68%, and 4.86% using the reference assay, with agreements higher than 99.32%. Conclusion: The results indicated that a rapid and accurate quadruplex RT-qPCR was developed for the detection and differentiation of CCoV, CRV, CPV, and CDV.

Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV) are important pathogens for gastroenteritis, which is characterized by vomiting, diarrhea, and dehydration. Four pairs of primers and probes were designed to target the FeKoV VP1, FeAstV ORF2, FeBuV VP2, and FRV NSP4 genes, and a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay capable of the simultaneous detection of four feline enteroviruses was developed after optimization of reaction conditions. The established quadruplex RT-qPCR assay showed high specificity, sensitivity, and reproducibility. The assay could detect and discriminate FeKoV, FeAstV, FeBuV, and FRV, but not other feline-related pathogens. The limits of detection (LODs) of FeKoV, FeAstV, FeBuV, and FRV were 109.761, 115.834, 125.481, and 113.875 copies/reaction, respectively. The intra- and inter-assay coefficients of variation (CV) were 0.15–1.61% and 0.15–1.59%, respectively. In all, 1869 clinical samples from Guangxi province in Southern China were tested using the developed assay, and the positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93%, 9.36%, 0.32%, and 0.75%, respectively. These samples were also tested using reference assays, and the coincidence rates of the results between the developed and reference methods were 99.63% (FeKoV), 98.72% (FeAstV), 100% (FeBuV), and 100% (FRV), respectively. The results indicated that the developed assay could provide a new detection method for these four viruses associated with feline gastroenteritis.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is the only known porcine neurotropic coronavirus, which is prevalent worldwide at present. It is of great significance to understand the genetic and evolutionary characteristics of PHEV in order to perform effective measures for prevention and control of this disease. In this study, a total of 6,986 tissue samples and nasopharyngeal swabs were collected from different regions of Guangxi province in southern China during 2021-2024, and were tested for PHEV using a quadruplex RT-qPCR. The positivity rate of PHEV was 2.81% (196/6,986), of which tissue samples and nasopharyngeal swabs had 2.05% (87/4,246) and 3.98% (109/2,740) positivity rates, respectively. Fifty PHEV positive samples were selected for PCR amplification and gene sequencing. Sequence analysis revealed that the nucleotide homology and amino acid similarities of S, M, and N genes were 94.3%-99.3% and 92.3%-99.2%, 95.0%-99.7% and 94.7%-100.0%, 94.0%-99.5% and 93.5%-99.3%, respectively, indicating M and N genes were more conservative than S gene. Phylogenetic trees based on these three genes revealed that PHEV strains from different countries could be divided into two groups G1 and G2, and the PHEV strains from Guangxi province obtained in this study distributed in subgroups G1c and G2b. Bayesian analysis revealed that the population size of PHEV has been in a relatively stable state since its discovery until it expanded sharply around 2015, and still on the slow rise thereafter. S gene sequences analysis indicated that PHEV strains existed variation of mutation, and recombination. The results indicated that the prevalent PHEV strains in Guangxi province had complex evolutionary trajectories and high genetic diversity. To the best of our knowledge, this is the first report on the genetic and evolutionary characteristics of PHEV in southern China.

African swine fever (ASF) is a highly contagious and lethal viral disease that causes severe hemorrhagic fever in pigs. It keeps spreading around the world, posing a severe socioeconomic risk and endangering biodiversity and domestic food security. ASF first outbroke in China in 2018, and has spread to most provinces nationwide. Genotypes I and II ASF virus (ASFV) as the etiological pathogens have been found in China. In this study, three pairs of specific primers and probes targeting the ASFV B646L gene, F1055L gene, and E183L gene were designed to detect universal, genotype I, and genotype II strains, respectively. A triplex crystal digital PCR (cdPCR) was established on the basis of optimizing various reaction conditions. The assay demonstrated remarkably sensitive with low limits of detection (LODs) of 5.120, 4.218, 4.588 copies/reaction for B646L, F1055L, and E183L gene, respectively; excellent repeatability with 1.24-2.01% intra-assay coefficients of variation (CVs) and 1.32-2.53% inter-assay CVs; good specificity for only detection of genotypes I and II ASFV, without cross-reactivity with PCV2, PRV, SIV, PRRSV, PEDV, FMDV, and CSFV. The triplex cdPCR was used to test 1,275 clinical samples from Guangxi province of China, and the positivity rates were 5.05, 3.22, and 1.02% for genotype I, genotype II, and co-infection of genotypes I and II, respectively. These 1,275 clinical samples were also detected using a reported reference triplex real-time quantitative PCR (qPCR), and the agreements of detection results between these two methods were more than 98.98%. In conclusion, the developed triplex cdPCR could be used as a rapid, sensitive, and accurate method to detect and differentiate genotypes I and II strains of ASFV.

Feline coronavirus (FCoV), as one of the important pathogens of feline viral gastroenteritis, has been attracting great attention. A total of 1869 rectal and nasal swabs, feces, and ascites samples were collected from eight regions in Guangxi province during 2021–2024. The multiplex RT-qPCR established in our laboratory was used to test these samples for FCoV, and 17.66% (330/1869) of the samples were positive for FCoV. The S, M, and N genes of 63 FCoV-positive samples were amplified and sequenced, and the genetic and evolutionary characteristics were analyzed. Similarity analysis showed that the nucleotide and amino acid homologies of S, M, and N genes were 81.2–99.6% and 70.2–99.5%, 89.9–100% and 91.6–100%, and 90.1–100% and 91.5–100%, respectively. Phylogenetic analysis revealed that all 63 FCoV strains, based on S gene sequences, belonged to type I FCoV (FCoV-I), and were clustered with Chinese strains and the Netherlands UU strains. Recombinant signals were detected in the S gene of strains GXLZ03-2022, GXLZ08-2022, and CCoV GD/2020/X9. The results suggest that FCoV is still prevalent in the Guangxi province of southern China, and the prevalent FCoV strains show high genetic diversity and novel epidemic characteristics.

Canine coronavirus (CCoV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAV-2), and canine norovirus (CNV) are important pathogens for canine viral gastrointestinal and respiratory diseases. Especially, co-infections with these viruses exacerbate the damages of diseases. In this study, four pairs of primers and probes were designed to specifically amplify the conserved regions of the CCoV M gene, CRCoV N gene, CAV-2 hexon gene, and CNV RdRp gene. After optimizing different reaction conditions, a quadruplex RT-qPCR was established for the detection of CCoV, CRCoV, CAV-2, and CNV. The specificity, sensitivity, and repeatability of the established assay were evaluated. Then, the assay was used to test 1688 clinical samples from pet hospitals in Guangxi province of China during 2022–2024 to validate its clinical applicability. In addition, these samples were also assessed using the reported reference RT-qPCR assays, and the agreements between the developed and reference assays were determined. The results indicated that the quadruplex RT-qPCR could specifically test only CCoV, CRCoV, CAV-2, and CNV, without cross-reaction with other canine viruses. The assay had high sensitivity with limits of detection (LODs) of 1.0 × 102 copies/reaction for CCoV, CRCoV, CAV-2, and CNV. The repeatability was excellent, with intra-assay variability of 0.19–1.31% and inter-assay variability of 0.10–0.88%. The positivity rates of CCoV, CRCoV, CAV-2, and CNV using the developed assay were 8.59% (145/1688), 8.65% (146/1688), 2.84% (48/1688), and 1.30% (22/1688), respectively, while the positivity rates using the reference assays were 8.47% (143/1688), 8.53% (144/1688), 2.78% (47/1688), and 1.24% (21/1688), respectively, with agreements of more than 99.53% between two methods. In conclusion, a quadruplex RT-qPCR with high sensitivity, specificity, and repeatability was developed for rapid, and accurate detection of CCoV, CRCoV, CAV-2, and CNV.

为了研究德保猪的繁殖性能,试验采集了 75,82,113,115,120日龄德保母猪卵巢,利用石蜡切片方法对德保母猪的初情期进行研究;选择8头后备德保母猪和9头经产德保母猪对其发情周期进行研究;统计20头初产德保母猪和5头经产德保母猪的产仔数据,计算母猪窝平均产仔数、仔猪平均出生重、公母比例(公:母)、出生存活率和35 d断奶存活率;观察14头德保小公猪的爬跨行为对其初次爬跨行为进行研究;采集 15,26,28,30,32,34,36,38,40,42,43,44,45,46,47,49,51,53,55,62,70日龄德保公猪睾丸,利用石蜡切片方法对德保公猪睾丸初次产生精子的日龄进行了研究.结果表明:德保母猪的初情期在120日龄左右,后备和经产德保母猪发情周期分别为(26.00±2.00)d和(23.89±1.97)d,差异显著(P＜0.05);初产德保母猪的窝平均产仔数为(7.45±1.80)头,显著小于经产德保母猪[(10.00±2.10)头,P＜0.05],但初产德保母猪的仔猪平均出生重、公母比例(公:母)、出生存活率、35 d断奶存活率与经产德保母猪差异不显著(P＞0.05);德保小公猪最早出现爬跨的日龄是8日龄;德保公猪睾丸初次产生精子的日龄是45日龄.说明德保母猪具有发情周期长的特点,德保公猪是较早出现爬跨行为、曲细精管较早出现精子的品种之一,具有明显的性早熟特征.

Rotavirus A species (RVA), RVB, RVC, and RVH are four species of rotaviruses (RVs) that are prevalent in pig herds, and co-infections occur frequently. In this study, a quadruplex real-time quantitative RT-PCR (RT-qPCR) for the simultaneous detection of four porcine RVs was developed by designing specific primers and probes based on the VP6 gene of RVA, RVB, RVC, and RVH, respectively. The method showed high specificity and could only detect RVA, RVB, RVC, and RVH, without cross-reaction with other porcine viruses; showed excellent sensitivity, with a limit of detection (LOD) of 1.5 copies/µL for each virus; showed good repeatability, with intra-assay coefficients of variation (CVs) of 0.15–1.14% and inter-assay CVs of 0.07–0.96%. A total of 1447 clinical fecal samples from Guangxi province in China were tested using the developed quadruplex RT-qPCR. The results showed that RVA (42.71%, 618/1447), RVB (26.95%, 390/1447), RVC (42.92%, 621/1447), and RVH (13.68%, 198/1447) were simultaneously circulating in the pig herds, and the co-infection rate of different species of rotaviruses was found to be up to 44.01% (579/1447). The clinical samples were also detected using one previously reported method, and the coincidence rate of the detection results using two methods was more than 99.65%. The phylogenetic tree based on the VP6 gene sequences of RVH revealed that the porcine RVH strains from Guangxi province belonged to the genotype I5, which was closely related to Japanese and Vietnamese strains. In summary, an efficient, sensitive, and accurate method for the detection and differentiation of RVA, RVB, RVC, and RVH was developed and applied to investigate the prevalence of porcine RVs in Guangxi province, China. This study is the first to report the prevalence of porcine RVH in China.

African swine fever virus (ASFV) was first identified in 1921 and is extensively prevalent around the world nowadays, which has a significant negative impact on the swine industry. In China, genotype II ASFV was first discovered in 2018, and has spread quickly to different provinces in a very short time; genotype I ASFV was first found in 2020, and has been reported in several provinces since then. To establish an accurate method for detection and differentiation of genotypes I and II ASFV, three primers and probes were designed targeting the ASFV B646L gene for different genotypes, the F1055L gene for genotype I, and the E183L gene for genotype II, and a triplex real-time quantitative PCR (qPCR) for differential detection of genotypes I and II ASFV was developed after optimizing the reaction conditions. The assay showed high sensitivity, and the limits of detection (LOD) of the B646L, F1055L, and E183L genes were 399.647 copies/reaction, 374.409 copies/reaction, and 355.083 copies/reaction, respectively; the coefficients of variation (CVs) of the intra-assay and the inter-assay were 0.22-1.88% and 0.16-1.68%, respectively, showing that this method had good repeatability; the assay could detect only ASFV, without cross-reactivity with other swine viruses including PRRSV, PEDV, PDCoV, CSFV, PRV, and PCV2, showing excellent specificity of this method. A total of 3,519 clinical samples from Guangxi province, southern China, were tested by the developed assay, and 8.16% (287/3,519) samples were found to be positive for ASFV, of which 0.17% (6/3,519) samples were positive for genotype I, 7.19% (253/3,519) samples for genotype II, and 0.80% (28/3,519) samples for genotypes I and II. At the same time, these clinical samples were also tested by a previously reported multiplex qPCR, and the agreement between these two methods was more than 99.94%. In summary, the developed triplex qPCR provided a fast, specific and accurate method for detection and differentiation of genotypes I and II ASFV.

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV) cause similar neurological symptoms in the infected pigs, and their differential diagnosis depends on laboratory testing. Four pairs of specific primers and probes were designed targeting the PHEV N gene, PRV gB gene, CSFV 5′ untranslated region (5’UTR), and JEV NS1 gene, respectively, and a quadruplex real-time quantitative RT-PCR (qRT-PCR) was developed to detect and differentiate PHEV, PRV, CSFV, and JEV. The assay showed high sensitivity, with the limit of detection (LOD) of 1.5 × 101 copies/μL for each pathogen. The assay specifically detected only PHEV, PRV, CSFV, and JEV, without cross-reaction with other swine viruses. The coefficients of variation (CVs) of the intra-assay and the inter-assay were less than 1.84%, with great repeatability. A total of 1,977 clinical samples, including tissue samples, and whole blood samples collected from Guangxi province in China, were tested by the developed quadruplex qRT-PCR, and the positivity rates of PHEV, PRV, CSFV, and JEV were 1.57% (31/1,977), 0.35% (7/1,977), 1.06% (21/1,977), and 0.10% (2/1,977), respectively. These 1,977 samples were also tested by the previously reported qRT-PCR assays, and the coincidence rates of these methods were more than 99.90%. The developed assay is demonstrated to be rapid, sensitive, and accurate for detection and differentiation of PHEV, PRV, CSFV, and JEV.

The nuclear pore complex (NPC) is the bidirectional gate that mediates the exchange of macromolecules or their assemblies between nucleus and cytoplasm 1 – 3 . The assembly intermediates of the ribosomal subunits, pre-60S and pre-40S particles, are among the largest cargoes of the NPC and the export of these gigantic ribonucleoproteins requires numerous export factors 4 , 5 . Here we report the cryo-electron microscopy structure of native pre-60S particles trapped in the channel of yeast NPCs. In addition to known assembly factors, multiple factors with export functions are also included in the structure. These factors in general bind to either the flexible regions or subunit interface of the pre-60S particle, and virtually form many anchor sites for NPC binding. Through interactions with phenylalanine-glycine (FG) repeats from various nucleoporins of NPC, these factors collectively facilitate the passage of the pre-60S particle through the central FG repeat network of the NPC. Moreover, in silico analysis of the axial and radial distribution of pre-60S particles within the NPC shows that a single NPC can take up to four pre-60S particles simultaneously, and pre-60S particles are enriched in the inner ring regions close to the wall of the NPC with the solvent-exposed surface facing the centre of the nuclear pore. Our data suggest a translocation model for the export of pre-60S particles through the NPC.

Duck Tembusu virus (DTMUV), duck circovirus (DuCV), and new duck reovirus (NDRV) have seriously hindered the development of the poultry industry in China. To detect the three pathogens simultaneously, a multiplex digital PCR (dPCR) was developed and compared with multiplex qPCR in this study. The multiplex dPCR was able to specifically detect DTMUV, DuCV, and NDRV but not amplify Muscovy duck reovirus (MDRV), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), H4 avian influenza virus (H4 AIV), H6 avian influenza virus (H6 AIV), and Newcastle disease virus (NDV). The standard curves showed excellent linearity in multiplex dPCR and qPCR and were positively correlated. The sensitivity results showed that the lowest detection limit of multiplex dPCR was 1.3 copies/μL, which was 10 times higher than that of multiplex qPCR. The reproducibility results showed that the intra- and interassay coefficients of variation were 0.06-1.94%. A total of 173 clinical samples were tested to assess the usefulness of the method; the positive detection rates for DTMUV, DuCV, and NDRV were 18.5, 29.5, and 14.5%, respectively, which were approximately 4% higher than those of multiplex qPCR, and the kappa values for the clinical detection results of multiplex dPCR and qPCR were 0.85, 0.89, and 0.86, indicating that the two methods were in excellent agreement.

推进高校课程思政建设,发挥好课程的育人作用,是提高人才培养质量的必备内容.广西大学动物科学专业《动物解剖与组织胚胎学》课程顺应国家战略,积极进行课程思政建设,形成了一些特色做法.作者就课程思政建设意识和能力的提升、课程思政内容供给的优化、课程思政教学方法手段及课程考核模式的创新、课程思政教学效果评价体系的建立与健全、具有广西大学特色的课程思政示范课程的打造等5个方面进行了总结,供读者们参考.