Molecular detection and characterization of Coxiella burnetii in aborted samples of livestock in China.

Coxiella burnetii is the causative agent of zoonotic Q fever. Animals are the natural reservoirs of C. burnetii, and domestic livestock represent the major sources of human infection. C. burnetii infection in pregnant females may causes abortion during late pregnancy, whereby massive shedding of C. burnetii with abortion products becomes aerosolized and persists in the environment. Therefore, monitoring and surveillance of this infection in livestock is important for the prevention of the C. burnetii transmission. Previous serological surveys have shown that C. burnetii infection is endemic in livestock in China. However, few data are available on the diagnosis of C. burnetii as a cause of abortion by molecular methods in livestock. To get a better understanding of the impact of C. burnetii infection on domestic livestock in China, a real-time PCR investigation was carried out on collected samples from different domestic livestock suffering abortion during 2021-2023. A total of 338 samples collected from eight herds of five livestock species were elected. The results showed that 223 (66 %) of the collected samples were positive for C. burnetii DNA using real-time PCR. For the aborted samples, 82 % (128/15) of sheep, 81 % (34/42) of goats, 44 % (15/34) of cattle, 69 % (18/26) of camels, and 50 % (17/34) of donkeys were positive for C. burnetii. Besides, 44 % (8/18) and 4 % (1/25) of asymptomatic individuals of sheep and donkey were also positive for C. burnetii. In addition, the positive samples were further confirmed by amplification and sequencing of the C. burnetii-specific isocitrate dehydrogenase (icd) gene. Phylogenetic analysis based on specific gene fragments of icd genes revealed that the obtained sequences in this study were clustered into two different groups associated with different origin of hosts and geographic regions. This is the first report confirming that C. burnetii exists in aborted samples of sheep, goats, cattle, donkeys and camels in China. Further studies are needed to fully elucidate the epidemiology of this pathogen in livestock as well as the potential risks to public health.

Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus.

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression.IMPORTANCEThe role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

Single-cell profiling of African swine fever virus disease in the pig spleen reveals viral and host dynamics.

African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.

The whole genomic analysis of the Orf virus strains ORFV-SC and ORFV-SC1 from the Sichuan province and their weak pathological response in rabbits.

The Orf virus (ORFV) is a member of the Parapoxvirus genus of the Poxviridae family and can cause contagious diseases in sheep, goats, and wild ungulates. In the present study, two ORFV isolates (ORFV-SC isolated from Sichuan province and ORFV-SC1 produced by 60 passages of ORFV-SC in cells) were sequenced and compared to multiple ORFVs. The two ORFV sequences had entire genome sizes of 14,0707 bp and 141,154 bp, respectively, containing 130 and 131 genes, with a G + C content of 63% for the ORFV-SC sequence and 63.9% for the ORFV-SC1 sequence. Alignment of ORFV-SC and ORFV-SC1 with five other ORFV isolates revealed that ORFV-SC, ORFV-SC1, and NA1/11 shared > 95% nucleotide identity with 109 genes. Five genes (ORF007, ORF20, ORF080, ORF112, ORF116) have low amino acids identity between ORFV-SC and ORFV-SC1. Mutations in amino acids result in changes in the secondary and tertiary structure of ORF007, ORF020, and ORF112 proteins. The phylogenetic tree based on the complete genome sequence and 37 single genes revealed that the two ORFV isolates originated from sheep. Finally, animal experiments demonstrated that ORFV-SC1 is less harmful to rabbits than ORFV-SC. The exploration of two full-length viral genome sequences provides valuable information in ORFV biology and epidemiology research. Furthermore, ORFV-SC1 demonstrated an acceptable safety profile following animal vaccination, indicating its potential as a live ORFV vaccine.

Generation and application of immortalized sertoli cell line from sheep testis.

Primary sheep testicular Sertoli cells (STSCs) are ideal for investigating the molecular and pathogenic processes of capripoxvirus. However, the high cost of isolation and culture of primary STSCs, time-consuming operation, and short lifespan greatly limit their real-world application. In our study, the primary STSCs were isolated and immortalized by transfection of a lentiviral recombinant plasmid containing simian virus 40 (SV40) large T antigen. Androgen-binding protein (ABP) and vimentin (VIM) protein expression, SV40 large T antigen activity, proliferation assays, and apoptosis analysis results showed that immortalized large T antigen STSCs (TSTSCs) still had the same physiological characteristics and biological functions as primary STSCs. Moreover, immortalized TSTSCs had strong anti-apoptosis ability, extended lifespan, and enhanced proliferative activity compared to primary STSCs, which had not transformed in vitro and showed any signs of malignancy phenotype in nude mice. Besides, immortalized TSTSCs were susceptible to goatpox virus (GTPV), lumpy skin disease virus (LSDV), and Orf virus (ORFV). In conclusion, immortalized TSTSCs are useful in vitro models to study GTPV, LSDV, and ORFV in a wide range of ways, suggesting that it can be safely used in virus isolation, vaccine and drug screening studies in future.

Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release.

Cyclic GMP-AMP synthase (cGAS) plays a key role in the innate immune responses to both DNA and RNA virus infection. Here, we found that enterovirus 71 (EV-A71), Seneca Valley virus (SVV), and foot-and-mouth disease virus (FMDV) infection triggered mitochondria damage and mitochondrial DNA (mtDNA) release in vitro and vivo. These responses were mediated by picornavirus 2B proteins which induced mtDNA release during viral replication. SVV infection caused the opening of mitochondrial permeability transition pore (mPTP) and led to voltage-dependent anion channel 1 (VDAC1)- and BCL2 antagonist/killer 1 (Bak) and Bak/BCL2-associated X (Bax)-dependent mtDNA leakage into the cytoplasm, while EV-A71 and FMDV infection induced mPTP opening and resulted in VDAC1-dependent mtDNA release. The released mtDNA bound to cGAS and activated cGAS-mediated antiviral immune response. cGAS was essential for inhibiting EV-A71, SVV, and FMDV replication by regulation of IFN-ß production. cGAS deficiency contributed to higher mortality of EV-A71- or FMDV-infected mice. In addition, we found that SVV 2C protein was responsible for decreasing cGAS expression through the autophagy pathway. The 9th and 153rd amino acid sites in 2C were critical for induction of cGAS degradation. Furthermore, we also show that EV-A71, CA16, and EMCV 2C antagonize the cGAS-stimulator of interferon genes (STING) pathway through interaction with STING, and highly conserved amino acids Y155 and S156 were critical for this inhibitory effect. In conclusion, these data reveal novel mechanisms of picornaviruses to block the antiviral effect mediated by the cGAS-STING signaling pathway, which will provide insights for developing antiviral strategies against picornaviruses.

Ocular Pharmacokinetic Properties of Intravitreally Injected Aflibercept in Rabbits After Using Brinzolamide/Timolol Eye Drops.

Purpose: To investigate the ocular pharmacokinetic properties of intravitreally injected aflibercept in rabbits after using brinzolamide 1%/timolol maleate 0.5% fixed-combination eye drops. Methods: The right eye of 5 rabbits was topically administered 30 µL of brinzolamide and timolol maleate eye drops twice a day (q12h). The 2 eyes of each rabbit were injected with 1.0 mg (0.025 cc) of aflibercept on the 2nd day after instilling the eye drops. The intraocular pressure of the rabbits was measured before injection and sampling. The aqueous humor was drawn at 1, 3, 7, 14, 21, and 28 days. Aflibercept concentrations in aqueous humor and vitreous humor (28 days) were measured by enzyme-linked immunosorbent assay. Results: The aflibercept aqueous concentrations in the right eye at days 7, 14, 21, and 28 after injection were all significantly higher than those in the left eye (P > 0.05, n = 5). The peak aqueous concentrations of aflibercept in right eyes (49.5 µg/mL) and left eyes (50.9 µg/mL) were both observed at 1 day after injection. The elimination half-life of aflibercept in the aqueous humor of the right eye (4.70 days) was 1 day longer than that of the left eye (3.65 days). The average percentage of residual aflibercept in the vitreous humor of the right eye (3.35%) was also significantly higher than that of the left eye (0.63%). Conclusions: Brinzolamide 1%/timolol maleate 0.5% fixed-combination eye drops can significantly extend the ocular residence time of intravitreally injected aflibercept.

Photo-induced cascaded harmonic and comb generation in silicon nitride microresonators.

Silicon nitride (Si3N4) is an ever-maturing integrated platform for nonlinear optics but mostly considered for third-order [χ(3)] nonlinear interactions. Recently, second-order [χ(2)] nonlinearity was introduced into Si3N4 via the photogalvanic effect, resulting in the inscription of quasi-phase-matched χ(2) gratings. However, the full potential of the photogalvanic effect in microresonators remains largely unexplored for cascaded effects. Here, we report combined χ(2) and χ(3) nonlinear effects in a normal dispersion Si3N4 microresonator. We demonstrate that the photo-induced χ(2) grating also provides phase-matching for the sum-frequency generation process, enabling the initiation and successive switching of primary combs. In addition, the doubly resonant pump and second-harmonic fields allow for effective third-harmonic generation, where a secondary optically written χ(2) grating is identified. Last, we reach a broadband microcomb state evolved from the sum-frequency-coupled primary comb. These results expand the scope of cascaded effects in microresonators.

A photonic integrated continuous-travelling-wave parametric amplifier.

The ability to amplify optical signals is of pivotal importance across science and technology typically using rare-earth-doped fibres or gain media based on III-V semiconductors. A different physical process to amplify optical signals is to use the Kerr nonlinearity of optical fibres through parametric interactions1,2. Pioneering work demonstrated continuous-wave net-gain travelling-wave parametric amplification in fibres3, enabling, for example, phase-sensitive (that is, noiseless) amplification4, link span increase5, signal regeneration and nonlinear phase noise mitigation6. Despite great progress7-15, all photonic integrated circuit-based demonstrations of net parametric gain have necessitated pulsed lasers, limiting their practical use. Until now, only bulk micromachined periodically poled lithium niobate (PPLN) waveguide chips have achieved continuous-wave gain16,17, yet their integration with silicon-wafer-based photonic circuits has not been shown. Here we demonstrate a photonic-integrated-circuit-based travelling-wave optical parametric amplifier with net signal gain in the continuous-wave regime. Using ultralow-loss, dispersion-engineered, metre-long, Si3N4 photonic integrated circuits18 on a silicon chip of dimensions 5 × 5 mm2, we achieve a continuous parametric gain of 12 dB that exceeds both the on-chip optical propagation loss and fibre-chip-fibre coupling losses in the telecommunication C band. Our work demonstrates the potential of photonic-integrated-circuit-based parametric amplifiers that have lithographically controlled gain spectrum, compact footprint, resilience to optical feedback and quantum-limited performance, and can operate in the wavelength ranges from visible to mid-infrared and outside conventional rare-earth amplification bands.

Third-harmonic-assisted four-wave mixing in a chip-based microresonator frequency comb generation.

Microcombs generated in photonic integrated circuits can provide broadband and coherent optical frequency combs with a high repetition rate from microwave to terahertz. Coherent microcombs formed in normal group velocity dispersion microresonators usually have a flat-top temporal profile, called platicon. Here, we propose a novel scheme to generate platicon in Si3N4 microresonator with the assistance of third-harmonic generation. The nonlinear coupling between the fundamental and the third-harmonic waves that draws support from third-order sum/difference frequency generation provides a new mechanism to achieve the phase matching of four-wave mixing in normal dispersion microresonators. We show that single or multiple platicons can be obtained by changing the third-harmonic nonlinear coupling strength and phase matching condition for third-order sum/difference frequency generation. Our work provides a promising solution to facilitate coherent and visible microcomb generation in a pure χ(3) microresonator, which is potential for self-referencing combs and optical clock stabilization.

The endocytosis of foot-and mouth disease virus requires clathrin and caveolin and is dependent on the existence of Rab5 and Rab7 in CHO-677 cells.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that causes severe vesicular disease of cloven-hoofed animals. Various endocytosis mechanisms are involved in the entry of FMDV after binding to the integrin and heparan sulfate (HS) receptors. However, the mechanism of FMDV using other unknown receptors to enter the cells remains unclear. Here, we reported that the endocytosis and endosomal pathways are employed by FMDV to invade the Chinese hamster ovary cell line (CHO-677) without the integrin and HS receptors. We demonstrated that the internalization of FMDV into CHO-677 cells was abrogated by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. Knockdown of the clathrin heavy chain decreased the viral protein abundance. Incubation of the CHO-677 cells with the inhibitors of caveolae-mediated endocytosis or transfection by caveolin-1 siRNA also limited FMDV replication. In addition, we determined that the acidic environment and the existence of dynamin were essential for FMDV infection in CHO-677 cells. The endosomal proteins Rab5 (early endosome) and Rab7 (late endosome), but not Rab11 (recycling endosome), were utilized by FMDV during infection. These data provide a new entry model of FMDV by unknown receptors which will help to better understand the pathogenesis mediated by FMDV.

A Naked-Eye Visual Reverse Transcription Loop-Mediated Isothermal Amplification with Sharp Color Changes for Potential Pen-Side Test of Foot-and-Mouth Disease Virus.

Visual loop-mediated isothermal amplification (LAMP) is qualified to be applied in the field to detect pathogens due to its simplicity, rapidity and cost saving. However, the color changes in currently reported visual reverse transcription LAMP (RT-LAMP) for foot-and-mouth disease virus (FMDV) detection are not so obvious to the naked eye, so interpretation of results is troublesome. In this study, a new naked-eye visual RT-LAMP to detect all seven distinct serotypes of FMDV was established based on the 3D genes by using pH-sensitive neutral red as the indicator, rendering a sharp contrast of color changes between the negative (light orange) and the positive (pink). Analytical sensitivity tests showed that the detection limit of the visual RT-LAMP was 104 copies/µL while those were 103 and 104 copies/µL for the RT-qPCR and conventional RT-PCR methods, respectively. Specificity tests proved that the established visual RT-LAMP assay had no cross-reactivity with other common livestock viruses. Furthermore, the analysis of 59 clinical samples showed 98.31% and 100% concordance with the RT-qPCR and the RT-PCR, respectively. The pan-serotypic FMD visual RT-LAMP assay could be suitable for a pen-side test of all seven serotypes of FMDV because the results could be easily distinguished by the naked eye without the requirement of complicated instruments and professional technicians. Hence, the novel method may have a promising prospect in field tests which exert an important role in monitoring, preventing, and controlling FMD, especially in regions with no PCR or qPCR instrument available.

Low-noise frequency-agile photonic integrated lasers for coherent ranging.

Frequency modulated continuous wave laser ranging (FMCW LiDAR) enables distance mapping with simultaneous position and velocity information, is immune to stray light, can achieve long range, operate in the eye-safe region of 1550 nm and achieve high sensitivity. Despite its advantages, it is compounded by the simultaneous requirement of both narrow linewidth low noise lasers that can be precisely chirped. While integrated silicon-based lasers, compatible with wafer scale manufacturing in large volumes at low cost, have experienced major advances and are now employed on a commercial scale in data centers, and impressive progress has led to integrated lasers with (ultra) narrow sub-100 Hz-level intrinsic linewidth based on optical feedback from photonic circuits, these lasers presently lack fast nonthermal tuning, i.e. frequency agility as required for coherent ranging. Here, we demonstrate a hybrid photonic integrated laser that exhibits very narrow intrinsic linewidth of 25 Hz while offering linear, hysteresis-free, and mode-hop-free-tuning beyond 1 GHz with up to megahertz actuation bandwidth constituting 1.6 × 1015 Hz/s tuning speed. Our approach uses foundry-based technologies - ultralow-loss (1 dB/m) Si3N4 photonic microresonators, combined with aluminium nitride (AlN) or lead zirconium titanate (PZT) microelectromechanical systems (MEMS) based stress-optic actuation. Electrically driven low-phase-noise lasing is attained by self-injection locking of an Indium Phosphide (InP) laser chip and only limited by fundamental thermo-refractive noise at mid-range offsets. By utilizing difference-drive and apodization of the photonic chip to suppress mechanical vibrations of the chip, a flat actuation response up to 10 MHz is achieved. We leverage this capability to demonstrate a compact coherent LiDAR engine that can generate up to 800 kHz FMCW triangular optical chirp signals, requiring neither any active linearization nor predistortion compensation, and perform a 10 m optical ranging experiment, with a resolution of 12.5 cm. Our results constitute a photonic integrated laser system for scenarios where high compactness, fast frequency actuation, and high spectral purity are required.

A photonic integrated circuit-based erbium-doped amplifier.

Erbium-doped fiber amplifiers revolutionized long-haul optical communications and laser technology. Erbium ions could provide a basis for efficient optical amplification in photonic integrated circuits but their use remains impractical as a result of insufficient output power. We demonstrate a photonic integrated circuit-based erbium amplifier reaching 145 milliwatts of output power and more than 30 decibels of small-signal gain-on par with commercial fiber amplifiers and surpassing state-of-the-art III-V heterogeneously integrated semiconductor amplifiers. We apply ion implantation to ultralow-loss silicon nitride (Si3N4) photonic integrated circuits, which are able to increase the soliton microcomb output power by 100 times, achieving power requirements for low-noise photonic microwave generation and wavelength-division multiplexing optical communications. Endowing Si3N4 photonic integrated circuits with gain enables the miniaturization of various fiber-based devices such as high-pulse-energy femtosecond mode-locked lasers.

Peste Des Petits Ruminants Virus N Protein Is a Critical Proinflammation Factor That Promotes MyD88 and NLRP3 Complex Assembly.

Inflammatory responses play a central role in host defense against invading pathogens. Peste des petits ruminants virus (PPRV) causes highly contagious acute or subacute disease of small ruminants. However, the precise mechanism by which PPRV regulates inflammatory responses remains unknown. Here, we revealed a novel mechanism by which PPRV induces inflammation. Our study showed that PPRV induced the secretion of interleukin 1ß (IL-1ß) by activating the NF-κB signaling pathway and the NLRP3 inflammasome. Moreover, PPRV replication and protein synthesis were essential for NLRP3 inflammasome activation. Importantly, PPRV N protein promoted NF-κB signaling pathway and NLRP3 inflammasome via direct binding of MyD88 and NLPR3, respectively, and induced caspase-1 cleavage and IL-1ß maturation. Biochemically, N protein interacted with MyD88 to potentiate the assembly of MyD88 complex and interacted with NLPR3 to facilitate NLRP3 inflammasome complex assembly by forming an N-NLRP3-ASC ring-like structure, leading to IL-1ß secretion. These findings demonstrate a new function of PPRV N protein as an important proinflammation factor and identify a novel underlying mechanism modulating inflammasome assembly and function induced by PPRV. IMPORTANCE An important part of the innate immune response is the activation of NF-κB signaling pathway and NLPR3 inflammasome, which is induced upon exposure to pathogens. Peste des petits ruminants virus (PPRV) is a highly contagious virus causing fever, stomatitis, and pneumoenteritis in goats by inducing many proinflammatory cytokines. Although the NF-κB signaling pathway and NLRP3 inflammasome play an important role in regulating host immunity and viral infection, the precise mechanism by which PPRV regulates inflammatory responses remains unknown. This study demonstrates that PPRV induces inflammatory responses. Mechanistically, PPRV N protein facilitates the MyD88 complex assembly by directly binding to MyD88 and promotes the NLRP3 inflammasome complex assembly by directly binding to NLRP3 to form ring-like structures of N-NLRP3-ASC. These findings provide insights into the prevention and treatment of PPRV infection.

Platicon microcomb generation using laser self-injection locking.

The past decade has witnessed major advances in the development and system-level applications of photonic integrated microcombs, that are coherent, broadband optical frequency combs with repetition rates in the millimeter-wave to terahertz domain. Most of these advances are based on harnessing of dissipative Kerr solitons (DKS) in microresonators with anomalous group velocity dispersion (GVD). However, microcombs can also be generated with normal GVD using localized structures that are referred to as dark pulses, switching waves or platicons. Compared with DKS microcombs that require specific designs and fabrication techniques for dispersion engineering, platicon microcombs can be readily built using CMOS-compatible platforms such as thin-film (i.e., thickness below 300 nm) silicon nitride with normal GVD. Here, we use laser self-injection locking to demonstrate a fully integrated platicon microcomb operating at a microwave K-band repetition rate. A distributed feedback (DFB) laser edge-coupled to a Si3N4 chip is self-injection-locked to a high-Q ( > 107) microresonator with high confinement waveguides, and directly excites platicons without sophisticated active control. We demonstrate multi-platicon states and switching, perform optical feedback phase study and characterize the phase noise of the K-band platicon repetition rate and the pump laser. Laser self-injection-locked platicons could facilitate the wide adoption of microcombs as a building block in photonic integrated circuits via commercial foundry service.

A sensitive and rapid bioanalytical method for the quantitative determination of luliconazole in rabbit eye tissues using UPLC-MS/MS assay.

Luliconazole (LCZ) is a novel antifungal imidazole with broad-spectrum and high susceptibility of Aspergillus and Fusarium are the dominant species of fungal keratitis, may potentially be a new medical treatment option for ocular fungal infection. To evaluate LCZ distribution in ocular tissues after topical application for the development of ophthalmic delivery system, it is important to have a bioanalytical method for measuring the drug concentrations in different ocular tissues and aqueous humor (AH). A selective and sensitive ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantification of LCZ in rabbit ocular tissues, including conjunctiva, cornea, AH, iris, lens, vitreous humor (VH), retinal choroid and sclera, using lanoconazole as internal standard (IS). Chromatographic separation was achieved on a Xterra MS, C18 column (2.1 × 50 mm, 3.5 µm) using mobile phase with formic acid solution (0.2%, v/v): acetonitrile (50:50, v/v) at a flow rate of 0.2 ml/min, and the run time was 2.5 min. Detection was performed using the transitions 354.1 → 150.3 m/z for LCZ and 320.1 → 150.3 m/z for IS by positive ion electrospray ionization in multiple reaction monitoring (MRM) mode. Method validation was conducted in accordance with U.S. Food and Drug Administration's regulatory guidelines for bioanalytical method validation. The calibration curves were linear over the concentration range from 2.80 ng/ml to 2038 ng/ml for conjunctiva, cornea and sclera, 2.09 ng/ml to 1019 ng/ml for AH, 2.09 ng/ml to 509.5 ng/ml for iris, 2.09 ng/ml to 203.8 ng/ml for retinal choroid and VH, 2.04 ng/ml to 101.9 ng/ml for lens, with all the squared correlation coefficients (r2) more than 0.99. The accuracy of the method was within the acceptable limit of 89.34%∼112.78% at the lower limit of quantification and other concentrations, Inter-day and intra-day precision values, expressed in terms of RSD (%), in all tissues were within 15% at all concentrations. The mean recoveries of LCZ in rabbit ocular tissues was 84.85%∼100.52%. No interference was found due to matrix components. Luliconazole was stable during the stability studies, including autosampler stability, benchtop stability, freeze/thaw stability and long-term stability. The method was successfully applied to the ocular pharmacokinetic and tissues distribution studies of LCZ in rabbit after topical administration of LCZ ophthalmic drug delivery system.

MGF360-9L Is a Major Virulence Factor Associated with the African Swine Fever Virus by Antagonizing the JAK/STAT Signaling Pathway.

African swine fever (ASF)-an aggressive infectious disease caused by the African swine fever virus (ASFV)-is significantly unfavorable for swine production. ASFV has a complex structure and encodes 150-167 proteins; however, the function of most of these proteins is unknown. This study identified ASFV MGF360-9L as a negative regulator of the interferon (IFN)-ß signal. Further evidence showed that MGF360-9L interacts with signal transducer and activator of transcription (STAT) 1 and STAT2 and degrades STAT1 and STAT2 through apoptosis and ubiquitin-proteasome pathways, respectively. Subsequently, the activation of IFN-ß signaling was inhibited. Naturally isolated or genetically manipulated live attenuated viruses are known to protect against the virulent parental ASFV strains. Therefore, through homologous recombination, we deleted MGF360-9L from the virulent ASFV CN/GS/2018 strain to construct a recombinant strain, ASFV-Δ360-9L. Compared with the parent ASFV CN/GS/2018 strain, the replication level of ASFV-Δ360-9L decreased in primary porcine alveolar macrophage cultures at 24 h postinfection, but the difference is unlikely to be biologically relevant. Notably, ASFV-Δ360-9L was partially attenuated in pigs. To our knowledge, this study is the first to uncover the function of MGF360-9L during ASFV infection. MGF360-9L inhibits IFN-ß signaling through the targeted degradation of STAT1 and STAT2. Furthermore, MGF360-9L is a key virulence gene of ASFV. Our findings reveal a new mechanism by which ASFV inhibits host antiviral response; this might facilitate the development of live attenuated ASFV vaccines. IMPORTANCE African swine fever-an acute, febrile, hemorrhagic, highly contacting, and highly lethal disease caused by African swine fever virus (ASFV)-jeopardizes the global pig industry. Understanding the mechanism ASFV employs to evade host defense during infection is essential for developing targeted drugs and vaccines against ASFV. To our knowledge, this study identifies the mechanism of innate immunity against by MGF360-9L and the effect of MGF360-9L on ASFV pathogenicity. The results showed that MGF360-9L may help ASFV escape the host immunity by degrading STAT1 and STAT2 and thus inhibiting IFN-ß signaling. MGF360-9L is also an important virulence factor of ASFV. The deletion of MGF360-9L reduces ASFV virulence in pigs. This study explored a new mechanism of ASFV against innate immunity and identified a new ASFV virulence factor; these findings may guide the development of live attenuated ASFV vaccines.

Analysis and Sequence Alignment of Peste des Petits Ruminants Virus ChinaSX2020.

The peste des petits ruminants virus (PPRV) mainly infects goats and sheep and causes a highly contagious disease, PPR. Recently, a PPRV strain named ChinaSX2020 was isolated and confirmed following an indirect immunofluorescence assay and PCR using PPRV-specific antibody and primers, respectively. A sequencing of the ChinaSX2020 strain showed a genome length of 15,954 nucleotides. A phylogenetic tree analysis showed that the ChinaSX2020 genome was classified into lineage IV of the PRRV genotypes. The genome of the ChinaSX2020 strain was found to be closely related to PPRVs isolated in China between 2013 and 2014. These findings revealed that not a variety of PRRVs but similar PPRVs were continuously spreading and causing sporadic outbreaks in China.