Characterization of Pseudomonas aeruginosa bacteriophages and control hemorrhagic pneumonia on a mice model.

Pseudomonas aeruginosa is one of the most common pathogens causing hemorrhagic pneumonia in Chinese forest musk deer. Multidrug-resistant P. aeruginosa is frequently isolated from the lungs of affected musk deer in Shaanxi Province, China. With the increasing bacterial drug resistance, commonly used antibiotics have shown limited efficacy against drug-resistant P. aeruginosa. Therefore, phages have garnered attention as a promising alternative to antibiotics among researchers. In this study, phages vB_PaeP_YL1 and vB_PaeP_YL2 (respectively referred to as YL1 and YL2) were isolated from mixed sewage samples from a farm. YL1 and YL2 exhibit an icosahedral head and a non-contractile short tail, belonging to the Podoviridae family. Identification results demonstrate good tolerance to low temperatures and pH levels, with minimal variation in potency within 30 min of UV irradiation. The MOI for both YL1 and YL2 was 0.1, and their one-step growth curve latent periods were 10 min and 20 min, respectively. Moreover, both single phage and phage cocktail effectively inhibited the growth of the host bacteria in vitro, with the phage cocktail showing superior inhibitory effects compared to the single phage. YL1 and YL2 possess double-stranded DNA genomes, with YL1 having a genome size of 72,187 bp and a total G + C content of 55.02%, while YL2 has a genome size of 72,060 bp and a total G + C content of 54.98%. YL1 and YL2 are predicted to have 93 and 92 open reading frames (ORFs), respectively, and no ORFs related to drug resistance or lysogeny were found in both phages. Genome annotation and phylogenetic analysis revealed that YL1 is closely related to vB_PaeP_FBPa1 (ON857943), while YL2 is closely related to vB_PaeP_FBPa1 (ON857943) and Phage26 (NC041907). In a mouse model of hemorrhagic pneumonia, phage cocktail treatment showed better control of the disease and significantly reduced lung bacterial load compared to single phage treatment. Therefore, YL1 and YL2 have the potential for the prevention and treatment of multidrug-resistant P. aeruginosa infections.

Lineage 1 PRRSVs Infection Induces Hemorrhagic Injury in Intestines of Piglets: Effects on Complement and Coagulation Cascades.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly transmissible disease of significant concern in the pig industry. Previous studies have demonstrated that the XM-2020 strain (a lineage 1.8 PRRSV IA/2012/NADC30) can induce special hemorrhagic injury in the small intestines. However, the specific mechanism underlying this injurious effect remains incompletely understood. In this study, we examined the pathogenic properties of XM-2020 and YC-2020 strains (a lineage 1.5 PRRSV IA/2014/NADC34) in piglets. Animal pathogenic tests revealed that with either Lineage 1 PRRSVs strains XM-2020 or YC-2020 demonstrated pronounced intestinal hemorrhage and suppression of peripheral immunological organs, comparing to JXA1 infection. Transcriptome analysis of diseased small intestines unveiled that PRRSV infection stimulated oxidative and inflammatory reactions. Remarkably, we also observed activation of the complement system alongside a notable down-regulation of complement and coagulation cascade pathways in the Lineage 1 PRRSVs infection group. Based on these findings, we propose that the primary mechanism driving the hemorrhagic injury of the small intestine caused by Lineage 1 PRRSVs is the suppression of complement and coagulation cascades resulting from immunosuppression. This discovery deepens our understanding of the pathogenicity of PRRSV in the small intestine and provides promising ways out for the development of innovative strategies aimed at controlling PRRSV.

Comparing the molecular evolution and recombination patterns of predominant PRRSV-2 lineages co-circulating in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses widespread epidemics in swine herds, yet the drivers underlying lineage replacements/fitness dynamics remain unclear. To delineate the evolutionary trajectories of PRRSV-2 lineages prevalent in China, we performed a comprehensive longitudinal phylodynamic analysis of 822 viral sequences spanning 1991-2022. The objectives encompassed evaluating lineage dynamics, genetic diversity, recombination patterns and glycosylation profiles. A significant shift in the dominance of PRRSV-2 sub-lineages has been observed over the past 3 decades, transitioning from sub-lineage 8.7 to sub-lineage 1.8, followed by extensive diversification. The analysis revealed discordant recombination patterns between the two dominant viral sub-lineages 1.8 and 8.7, underscoring that modular genetic exchanges contribute significantly to their evolutionary shaping. Additionally, a strong association was found between recombination breakpoint locations and transcriptional regulatory sequences (TRSs). Glycosylation patterns also demonstrated considerable variability across sub-lineages and temporally, providing evidence for immune-driven viral evolution. Furthermore, we quantified different evolutionary rates across sub-lineages, with sub-lineage 1.8 uniquely displaying the highest nucleotide substitution rates. Taken together, these findings provide refined insight into the evolutionary mechanisms underpinning cyclic shifts in dominance among regionally circulating PRRSV sub-lineages.

Effects of a novel Bacillus subtilis GXYX crude lipopeptide against Salmonella enterica serovar Typhimurium infection in mice.

The increased rate of antibiotic resistance strongly limits the resolution of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Therefore, new strategies to control bacterial infections are urgently needed. Bacillus subtilis (B. subtilis) and its metabolites are desirable antibacterial agents. Here, we aimed to evaluate the antibacterial activity of the novel B. subtilis strain GXYX (No: PRJNA940956) crude lipopeptide against S. Typhimurium. In vitro, GXYX crude lipopeptides affected S. Typhimurium biofilm formation and swimming and attenuated the adhesion and invasion abilities of S. Typhimurium toward BHK-21 cells; in addition, it inhibited the mRNA expression of the filA, filC, csgA, and csgB genes, which are related to the adhesion and invasion ability of S. Typhimurium. In vivo, pretreatment with GXYX crude lipopeptide via intragastric administration improved the survival rate by 30%, which was related to reductions in organ bacterial loads and clinical signs in mice. Intragastric administration of GXYX crude lipopeptide significantly downregulated the mRNA levels of TNF-α, IL-1ß, IL-12 and IL-6 in response to S. Typhimurium-induced inflammation compared with intraperitoneal injection. Moreover, it significantly improved the intestinal barrier-related gene (ZO-1, claudin-1, occludin-1) mRNA levels in intestinal tissue damaged by S. Typhimurium infection. In conclusion, GXYX crude lipopeptides were effective at reducing S. Typhimurium colonization, laying a foundation for the further development of novel antibacterial agents.

Genetic and pathogenic characterization of an NADC-34-like isolate of porcine reproductive and respiratory syndrome virus.

In this study, an NADC34-like strain of porcine reproductive and respiratory syndrome virus (PRRSV), YC-2020, was isolated from a pig farm in Yuncheng, Shanxi Province, China. Phylogenetic and molecular evolutionary analysis showed that the genome sequence of YC-2020 was very similar to those of NADC34-like PRRSV strains in the ORF2-7 region. However, it was more closely related to NADC30-like PRRSV and highly pathogenic (HP) PRRSV in the NSP2 and NSP3-9 coding regions, respectively, suggesting that recombination had occurred between viruses belonging to lineages 1 and 8. Piglets infected with YC-2020 exhibited mild clinical signs, but they had severe histopathological lesions in their lungs. These findings reveal novel genetic and pathogenic features of this isolate.

γ-Cyclodextrin-based metal-organic frameworks for lactonic sophorolipid application in enhanced oil recovery.

Lactonic sophorolipid (LSL) exhibits numerous surfactant activities, such as emulsification, wetting action, dispersion effect, and oil-washing activities. Nevertheless, LSLs have poor water solubility, which restrains their application in the petroleum industry. In this research, a new compound, lactonic sophorolipid cyclodextrin metal-organic framework (LSL-CD-MOFs), was obtained by loading LSL into γ-cyclodextrin metal-organic frameworks (γ-CD-MOFs). The LSL-CD-MOFs were characterized by N2 adsorption analysis, X-ray powder diffraction analysis, Fourier transform infrared spectroscopy, and thermogravimetric analysis. Loading LSL into γ-CD-MOFs significantly increased the apparent water solubility of LSL. However, the critical micelle concentration of LSL-CD-MOFs was similar to that of LSL. Furthermore, LSL-CD-MOFs effectively reduced the viscosities and improved the emulsification indices of oil-water mixtures. Oil-washing tests, which were conducted using oil sands, revealed that the LSL-CD-MOFs yielded an oil-washing efficiency of 85.82 % ± 2.04 %. Overall, γ-CD-MOFs are promising carriers for LSL, and LSL-CD-MOFs are a potential, low-cost, new, green surfactant for enhanced oil recovery.

Electronic, Tensile, and Sliding Characteristics of the C/Ti Interface: A First-Principles Study.

In this paper, according to the C(111) surface and Ti(112̅0) surface relative positions, three stacking interface models were constructed by the first-principles method, and they were defined as 1st-C(111)/Ti(112̅0), 2nd-C(111)/Ti(112̅0), and 4th-C(111)/Ti(112̅0), respectively. After calculation, the work of interfacial adhesion of the 1st-C(111)/Ti(112̅0), 2nd-C(111)/Ti(112̅0), and 4th-C(111)/Ti(112̅0) interface models is found to be 9.689, 10.246, and 9.714 J/m2, respectively, and their interface energies are observed to be 1.064, 0.507, and 1.039 J/m2, respectively. Moreover, the electronic characteristics of C(111)/Ti(112̅0) interfaces are dominated by polar covalent bonds, supplemented by certain metallicity. When the strain reaches 13, 15, and 12%, respectively, the maximum tensile stress values of 1st-C(111)/Ti(112̅0), 2nd-C(111)/Ti(112̅0), and 4th-C(111)/Ti(112̅0) interface models are observed to be 16.207, 19.183, and 17.393 GPa, respectively. After all C(111)/Ti(112̅0) interfaces fracture under tension, the Ti atoms of the Ti(112̅0) surface are transferred to the C(111) surface, indicating that the strength of Ti-C bonds at the interface is higher than the strength of Ti-Ti bonds inside the Ti(112̅0) surface. The maximum value of the sliding potential energy surface is 1.709 J/m2; the maximum value of the potential energy curve is 0.445 J/m2; and the ideal shear strength of the C(111)/Ti(112̅0) interface is 0.386 GPa. In summary, the interfacial adhesion property of the 2nd-C(111)/Ti(112̅0) interface is better than those of 1st-C(111)/Ti(112̅0) and 4th-C(111)/Ti(112̅0) interfaces.

The transcriptional characteristics of NADC34-like PRRSV in porcine alveolar macrophages.

The widespread and endemic circulation of porcine reproductive and respiratory syndrome virus (PRRSV) cause persistent financial losses to the swine industry worldwide. In 2017, NADC34-like PRRSV-2 emerged in northeastern China and spread rapidly. The dynamics analysis of immune perturbations associated with novel PRRSV lineage is still incomplete. This study performed a time-course transcriptome sequencing of NADC34-like PRRSV strain YC-2020-infected porcine alveolar macrophages (PAMs) and compared them with JXA1-infected PAMs. The results illustrated dramatic changes in the host's differentially expressed genes (DEGs) presented at different timepoints after PRRSV infection, and the expression profile of YC-2020 group is distinct from that of JXA1 group. Functional enrichment analysis showed that the expression of many inflammatory cytokines was up-regulated following YC-2020 infection but at a significantly lower magnitude than JXA1 group, in line with the trends for most interferon-stimulated genes (ISGs) and their regulators. Meanwhile, numerous components of histocompatibility complex (MHC) class II and phagosome presented a stronger transcription suppression after the YC-2020 infection. All results imply that YC-2020 may induce milder inflammatory responses, weaker antiviral processes, and more severe disturbance of antigen processing and presentation compared with HP-PRRSV. Additionally, LAPTM4A, GLMP, and LITAF, which were selected from weighted gene co-expression network analysis (WGCNA), could significantly inhibit PRRSV proliferation. This study provides fundamental data for understanding the biological characteristics of NADC34-like PRRSV and new insights into PRRSV evolution and prevention.

A novel Pseudorabies virus vaccine developed using HDR-CRISPR/Cas9 induces strong humoral and cellular immune response in mice.

Outbreaks of Pseudorabies (PR) by numerous highly virulent and antigenic variant Pseudorabies virus (PRV) strains have been causing severe economic losses to the pig industry in China since 2011. However, current commercial vaccines are often unable to induce thorough protective immunity. In this study, a TK/gI/gE deleted recombinant PRV expressing GM-CSF was developed by using the HDR-CRISPR/Cas9 system. Here, a four-sgRNA along with the Cas9D10A targeting system was utilized for TK/gI/gE gene deletion and GM-CSF insertion. Our study showed that the four-sgRNA targeting system appeared to have higher knock-in efficiency for PRVs editing. The replication of the recombinant PRVs were slightly lower than that of the parental strain, but they appeared to have similar properties in terms of growth curves and plaque morphology. The mice vaccinated with the recombinant PRV expressing GM-CSF via intramuscular injection showed no obvious clinical symptoms, milder pathological lesions, and were completely protected against wild-type PRV challenge. When compared to the triple gene-deleted PRV, the gB antibodies and neutralizing antibody titers were improved and the immunized mice appeared to have lower viral load and higher mRNA levels of IL-2, IL-4, IL-6, and IFN-γ in spleens. Our study offers a novel approach for recombinant PRV construction, and the triple gene-deleted PRV expressing GM-CSF could serve as a promising vaccine candidate for PR control.

A NADC30-like PRRSV causes serious intestinal infections and tropism in piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic loss to China's swine industry. Currently, a novel type 2 PRRSV, called the NADC30-like strain, is epidemic in numerous provinces of China. In this study, a NADC30-Like PRRSV strain was isolated in primary alveolar macrophage (PAM) cells from fecal samples collected from a local pig farm, which suffered severe diarrhea. A pathogenicity comparison study was conducted in 6-week-old piglets by inoculating highly pathogenic HP-PRRSV and NADC30-Like PRRSV isolates. RT-qPCR revealed detection of NADC30-Like PRRSV but not the HP-PRRSV in the intestine. PRRSV infection-related lesions were observed in the intestine were further confirmed by histopathological and immunohistochemical examination (IHC). In addition, severe virus infections were also detected by RT-qPCR. Based on clinical observation and pathogenicity experiments, we confirmed that NADC30-Like PRRSV gained more tissue tropism, especially in the small intestine. This may be the one reason explaining why NADC-Like 30 PRRSV become a major epidemic strain in China since the first outbreak in 2013.

Pressure-induced structure, elasticity, intrinsic hardness and ideal strength of tetragonal C4N.

A tetragonal C4N (t-C4N) structure was predicted via CALYPSO code, and the effects of pressure on its structural and mechanical properties were studied. The results show that t-C4N is different from various 2D CxNy compounds with a new type 3D crystal structure, which is similar to diamond. Bulk t-C4N is equipped with excellent elastic properties. When the pressure is increased from 0 GPa to 350 GPa, its bulk modulus B, shear modulus G and Young's modulus E are increased from 426.9 GPa to 1123.1 GPa, 371.4 GPa to 582.9 GPa and 863.7 GPa to 1490.9 GPa, respectively. The anisotropic Bmax, Gmax and Emax are increased from 582.38 GPa to 1751.41 GPa, 478.29 GPa to 1033.97 GPa and 1281.26 GPa to 2490.14 GPa, respectively. When the pressure is 0 GPa, the hardness calculated by Chen's and Tian's models are 51.15 GPa and 51.81 GPa, respectively. Its ideal tensile strength in [111] orientation is the smallest (63.46 GPa), which indicates that the (111) planes allow easy cleavage. The smallest ideal shear strength (67.98 GPa) can be obtained in the (111)[11̄0] orientation, which suggests its theoretical hardness is about 67.98 GPa. Due to its excellent mechanical properties, t-C4N can be used as an industrial superhard material.

Nanopore-Based Direct RNA-Sequencing Reveals a High-Resolution Transcriptional Landscape of Porcine Reproductive and Respiratory Syndrome Virus.

The TRS-mediated discontinuous transcription process is a hallmark of Arteriviruses. Precise assessment of the intricate subgenomic RNA (sg mRNA) populations is required to understand the kinetics of viral transcription. It is difficult to reconstruct and comprehensively quantify splicing events using short-read sequencing, making the identification of transcription-regulatory sequences (TRS) particularly problematic. Here, we applied long-read direct RNA sequencing to characterize the recombined RNA molecules produced in porcine alveolar macrophages during early passage infection of porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequencing two PRRSV isolates, namely XM-2020 and GD, we revealed a high-resolution and diverse transcriptional landscape in PRRSV. The data revealed intriguing differences in subgenomic recombination types between the two PRRSVs while also demonstrating TRS-independent heterogeneous subpopulation not previously observed in Arteriviruses. We find that TRS usage is a regulated process and share the common preferred TRS in both strains. This study also identified a substantial number of TRS-mediated transcript variants, including alternative-sg mRNAs encoding the same annotated ORF, as well as putative sg mRNAs encoded nested internal ORFs, implying that the genetic information encoded in PRRSV may be more intensively expressed. Epigenetic modifications have emerged as an essential regulatory layer in gene expression. Here, we gained a deeper understanding of m5C modification in poly(A) RNA, elucidating a potential link between methylation and transcriptional regulation. Collectively, our findings provided meaningful insights for redefining the transcriptome complexity of PRRSV. This will assist in filling the research gaps and developing strategies for better control of the PRRS.

Effects of Ti Doping on Structure and Internal Stress of Amorphous Carbon Films on the γ-Fe Substrate: Molecular Dynamics Simulation.

In this paper, C and Ti were used as the deposition atoms in the molecular dynamics model. The effects of Ti doping percentages (2, 8, 14, and 20 atom %) on the structure and internal stress of a-C films were investigated. The results showed that with the increase in the Ti percentage, the density of the intrinsic zone is gradually increased, while the growth rate is slowly decreased. The internal stress is gradually changed from compressive stress to tensile stress. Among them, when the Ti percentage is 14 atom %, the internal stress is closer to 0. The C percentage with sp3 hybridization is decreased, while those with sp2 and sp hybridizations are increased. The positions of the first and second peaks in the RDF were shifted to the left. Moreover, the distribution of bond lengths and bond angles in the intrinsic zone tend to change from diamond to graphite, which proves that Ti doping leads to the graphitization of a-C films. In addition, Ti doping affects the internal stress of a-C films by changing the C percentage with sp3 hybridization in them.

High-Temperature Sliding Friction Behavior of Amorphous Carbon Films: Molecular Dynamics Simulation.

With the development of the aerospace industry, the requirement for mechanical parts, which are serviced under extreme conditions such as high temperature, is more and more severe. Amorphous carbon (a-C) films are widely used in the aviation field as a protective coating because of their excellent antiwear and friction-reduction properties. However, a-C films are vulnerable to failure in a high-temperature environment, and a series of complex changes in the friction process make it a challenge to put forward the friction mechanism. Here, the sliding friction behaviors of amorphous carbon (a-C) films at different simulated temperatures (STs) (300-1300 K) were analyzed by molecular dynamics. The density, average coordination number, and local residual stress as well as the hybridization of sp, sp2, and sp3 of a-C films were analyzed to reveal the high-temperature sliding friction mechanism of a-C films. The results show that the friction coefficient (µ) of a-C films increased with increase in ST. Meanwhile, the friction mechanisms of a-C films are different at an ST lower than 800 K and higher than 1100 K. Compared with those before sliding, the local residual stress of all a-C films is relaxed, which causes transformation of sp3 into sp2. Moreover, when ST is lower than 800 K, the µ increased with increase in sp3%. When ST is higher than 1100 K, the stability of a-C films is broken, which results in the rapid increase in µ.

Adjuvant Effects of Platycodin D on Immune Responses to Infectious Bronchitis Vaccine in Chickens.

Adjuvants are common vaccine components. Novel adjuvants may improve the protective immunity conferred by vaccines against poultry diseases. Here, a less-hemolytic saponin, platycodin D (PD), isolated from the root of Platycodon grandiflorum was investigated as a potential alternative adjuvant. PD was tested as an adjuvant in the infectious bronchitis (IB) vaccine, because the existing IB vaccine has often failed to induce effective immune responses. The adjuvant activity of PD in conjunction with IB vaccine was evaluated in this study. Compared to control treatment, PD treatment significantly increased the proliferation of chicken peripheral blood mononuclear cells, concentration of interferon-γ in culture supernatants, and anti-IB antibody titer. In chickens pre-challenged with the Mass 41 infectious bronchitis virus (IBV), PD administration resulted in fewer and less severe clinical signs, lower mortality rate, and higher protection compared to control treatment. Histopathological examination showed that the lungs and kidneys of PD-treated chickens displayed fewer pathological lesions than those of control chickens. Our results also demonstrated that this new vaccine adjuvant improved chicken humoral and cellular immune responses without any side effects. Hence, our findings suggest that PD might serve as an effective adjuvant in IBV vaccines.

Effect of the flavonoid baicalein as a feed additive on the growth performance, immunity, and antioxidant capacity of broiler chickens.

Baicalein, the main flavonoid extracted from the root of Scutellaria baicalensis Georgi, has been demonstrated to exert multiple pharmacological effects, and thus could be utilized as a potential feed additive in broiler chickens. This study evaluated the effects of broiler chicken diet supplementation with baicalein on growth performance, immunity, and antioxidant activity at levels of 100 and 200 mg/kg. No significant effect on average daily feed intake (P > 0.05) of broilers with diets supplemented with baicalein was found compared to those on the basal diet or butylated hydroxytoluene (BHT) during the 35-d feeding trial. The addition of baicalein to the basal diet significantly increased average body weight, body weight gain, average weight gain, and the feed conversion ratio of birds during 21 to 42 d and 7 to 42 d of age, respectively. The best numerical values for the overall growth performance were observed in broilers fed on diets containing 200 mg/kg of baicalein. Baicalein supplementation significantly increased the ratio of CD3+/CD4+ and CD3±/CD8+, the concentration of IFN-γ, anti-IB antibody titer, and the spleen index compared with the control group (P < 0.05). Total cholesterol, the ratio of non-HDL-C/HDL-C, LDL-C/HDL-C, TC/HDL-C, triglycerides, and low-density lipoprotein cholesterol were significantly decreased after intake of baicalein compared with both the basal diet and the BHT-supplemented diet, whereas the SOD, GSH-Px, and CAT activity in the serum increased with the supplementation of baicalein. The T-AOC activity, T-SOD, and GSH-Px level in liver tissues was significantly increased by inclusion of baicalein, and intake of baicalein or BHT significantly decreased the malondialdehyde level found both in serum and meat tissue. Thus, the results obtained here indicate that the baicalein can be used as an effective natural feed additive in broiler chicken diets, and that 100 to 200 mg/kg can be considered as the optimum dosage.

Growth characteristics of primary M7C3 carbide in hypereutectic Fe-Cr-C alloy.

The microstructure of the hypereutectic Fe-Cr-C alloy is observed by optical microscopy (OM). The initial growth morphology, the crystallographic structure, the semi-molten morphology and the stacking faults of the primary M7C3 carbide are observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The in-suit growth process of the primary M7C3 carbide was observed by confocal laser microscope (CLM). It is found that the primary M7C3 carbide in hypereutectic Fe-Cr-C alloy is irregular polygonal shape with several hollows in the center and gaps on the edge. Some primary M7C3 carbides are formed by layers of shell or/and consist of multiple parts. In the initial growth period, the primary M7C3 carbide forms protrusion parallel to {} crystal planes. The extending and revolving protrusion forms the carbide shell. The electron backscattered diffraction (EBSD) maps show that the primary M7C3 carbide consists of multiple parts. The semi-molten M7C3 carbide contains unmelted shell and several small-scale carbides inside, which further proves that the primary M7C3 carbide is not an overall block. It is believed that the coalescence of the primary M7C3 carbides is ascribed to the growing condition of the protrusion and the gap filling process.

Aerobactin synthesis genes iucA and iucC contribute to the pathogenicity of avian pathogenic Escherichia coli O2 strain E058.

Aerobactin genes are known to be present in virulent strains and absent from avirulent strains, but contributions of iucC and iucA, which are involved in aerobactin synthesis, to the pathogenicity of avian pathogenic Escherichia coli (APEC) have not been clarified. In this study, effects of double mutants (iucA/iutA or iucC/iutA) compared to those of single mutants (iucA, iucC or iutA) of aerobactin genes on the virulence of APEC strain E058 were examined both in vitro (aerobactin production, ingestion into HD-11 cells, survival in chicken serum) and in vivo (competitive growth against parental strain, colonization and persistence). In competitive co-infection assays, compared to the E058 parental strain, the E058ΔiucA mutant was significantly reduced in the liver, kidney, spleen (all P<0.01), heart and lung (both P<0.001). The E058ΔiutA mutant also was significantly reduced in the liver, lung, kidney (all P<0.01), heart and spleen (both P<0.001). The E058ΔiucC mutant was significantly attenuated in the heart and kidney (both P<0.05) and showed a remarkable reduction in the liver, spleen and lung (P<0.01); meanwhile, both E058ΔiucAΔiutA and E058ΔiucCΔiutA double mutants were sharply reduced as well (P<0.001). In colonization and persistence assays, compared with E058, recovered colonies of E058ΔiucA were significantly reduced from the lung, liver, spleen and kidney (P<0.01) and significantly reduced in the heart (P<0.001). E058ΔiutA was significantly reduced from the heart, lung, liver, spleen and kidney (P<0.01). E058ΔiucC, E058ΔiucAΔiutA and E058ΔiucCΔiutA were significantly decreased in all organs tested (P<0.001). These results suggest that iutA, iucA and iucC play important roles in the pathogenicity of APEC E058.

Construction of iucB and iucBiutA mutants of avian pathogenic Escherichia coli and evaluation of their pathogenicity.

Aerobactin counts for much to the pathogenesis of avian pathogenic Escherichia coli (APEC), iutA is responsible for the expression of a specific outer membrane receptor protein for ferric aerobactin, and iucB is involved in the aerobactin synthesis. To our knowledge, the contribution of iucB to the pathogenicity of APEC has not been assessed till now. In this study, the mutants E058ΔiucB and E058ΔiucBΔiutA were constructed and characterized. There were no differences observed in a series of tests including the embryo lethality, invasion assay in HD11 cells and the ability to survive in SPF chicken serum. Meanwhile, the mutants showed decreased pathogenicity as compared with the wild-type strain through a series of experiments in vivo. The mutants E058ΔiucB and E058ΔiucBΔiutA greatly reduced in all of the tested tissues in vivo persistence (p<0.001). In the meantime, the mutants had no ability to produce aerobactin. Reintroduction of the iucB gene on a multicopy expression plasmid pGEX-6p-1 restored the capacity to produce aerobactin as similar to that of wild-type strain E058. The results indicated that the iucB gene related virulence factors including the iron assimilation system were important for the pathogenesis of APEC E058. As showed in the in vivo competition assay, compared to the parental strain E058, E058ΔiucB had a significant reduction of bacterial loads in heart (p<0.01), liver (p<0.01), kidney (p<0.01), spleen (p<0.05) and lung (p<0.05), respectively, while E058ΔiucBΔiutA had a sharp reduction in all of the five tissues to be tested (p<0.001). These results suggested that the single gene either iucB or iutA was likely to be involved directly or indirectly in iron uptake for the pathogenicity of APEC E058, and there was an obviously synergistic effect between iucB and iutA genes on the pathogenicity of APEC E058.

Identification of B-cell epitopes in the NSP1 protein of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) was divided into North American and European genotypes. NSP1 was an important non-structural protein of PRRSV, which was auto-cleaved from the replicase polyprotein into NSP1α and NSP1ß subunits and played an important role in the immune suppression. In this study, six monoclonal antibodies (MAbs) against the recombinant PRRSV NSP1, expressed in Escherichia coli system, were screened out and identified. Western blot and IFA results indicated that 4 out of 6 MAbs recognized the recombinant NSP1α and 2 MAbs recognized NSP1ß. Epitope mapping results indicated that MAb 4H2 recognized the linear epitopes E(54)EPLRW(59) in NSP1α, MAbs (2G5, 3E11 and 4D4) recognized the epitopes H(157)VLTNLP(163) in NSP1α, and MAbs 3C7 and 1H7 reacted with the epitopes 185aa to 232aa in NSP1ß. Protein sequence alignment of NSP1 indicated E(54)EPLRW(59) was conserved in all North American PRRSV strains, whereas European type strains has variable amino acids in this region. The epitope H(157)VLTNLP(163) was relatively conserved among all PRRSV strains, except for a L162→S162 change in European type strains. The epitope 185-232aa was variable among North American PRRSV strains. These results may facilitate future investigations into the function of NSP1 of PRRSV and diagnostic methods for PRRSV infection.