Dual auxotrophy coupled red labeling strategy for efficient genome editing in Saccharomyces cerevisiae.

The homologous recombination strategy has a long history of editing Saccharomyces cerevisiae target genes. The application of CRISPR/Cas9 strategy to editing target genes in S. cerevisiae has also received a lot of attention in recent years. All findings seem to indicate that editing relevant target genes in S. cerevisiae is an extremely easy event. In this study, we systematically analyzed the advantages and disadvantages of homologous recombination (HR) strategy, CRISPR/Cas9 strategy, and CRISPR/Cas9 combined homology-mediated repair (CRISPR/Case9-HDR) strategy in knocking out BY4742 ade2. Our data showed that when the ade2 was knocked out by HR strategy, a large number of clones appeared to be off-target, and 10 %-80 % of the so-called knockout clones obtained were heteroclones. When the CRISPR/Cas9 strategy was applied, 60% of clones were off-target and the rest were all heteroclones. Interestingly, most of the cells were edited successfully, but at least 60 % of the clones were heteroclones, when the CRISPR/Cas9-HDR strategy was employed. Our results clearly showed that the emergence of heteroclone seems inevitable regardless of the strategies used for editing BY4742 ade2. Given the characteristics of BY4742 defective in ade2 showing red on the YPD plate, we attempted to build an efficient yeast gene editing strategy, in which the CRISPR/Cas9 combines homology-mediated repair template carrying an ade2 expression cassette, BY4742(ade2Δ0) as the start strain. We used this strategy to successfully achieve 100 % knockout efficiency of trp1, indicating that technical challenges of how to easily screen out pure knockout clones without color phenotype have been solved. Our data showed in this study not only establishes an efficient yeast gene knockout strategy with dual auxotrophy coupled red labeling but also provides new ideas and references for the knockout of target genes in the monokaryotic mycelium of macrofungi.

Water-Assisted Growth of Twisted 3R-Stacked MoSe2 Spirals and Its Dramatically Enhanced Second Harmonic Generations.

Enhanced second-harmonic generation (SHG) responses are reported in monolayer transition metal dichalcogenides (e.g., MX2 , M: Mo, W; X: S, Se) due to the broken symmetries. The 3R-like stacked MX2 spiral structures possessing the similar broken inversion symmetry should present dramatically enhanced SHG responses, thus providing great flexibility in designing miniaturized on-chip nonlinear optical devices. To achieve this, the first direct synthesis of twisted 3R-stacked chiral molybdenum diselenide (MoSe2 ) spiral structures with specific screw dislocations (SD) arms is reported, via designing a water-assisted chemical vapor transport (CVT) approach. The study also clarifies the formation mechanism of the MoSe2 spiral structures, by precisely regulating the precursor supply accompanying with multiscale characterizations. Significantly, an up to three orders of magnitude enhancement of the SHG responses in twisted 3R stacked MoSe2 spirals is demonstrated, which is proposed to arise from the synergistic effects of broken inversion symmetry, strong light-matter interaction, and band nesting effects. Briefly, the work provides an efficient synthetic route for achieving the 3R-stacked TMDCs spirals, which can serve as perfect platforms for promoting their applications in on-chip nonlinear optical devices.

Core root-associated prokaryotic community and its relationship to host traits across wheat varieties.

The root-associated microbiomes play important roles in plant growth. However, it is largely unknown how wheat variety evolutionary relatedness shapes each subcommunity in the root microbiome and, in turn, how these microbes affect wheat yield and quality. Here we studied the prokaryotic communities associated with the rhizosphere and root endosphere in 95 wheat varieties at regreening and heading stages. The results indicated that the less diverse but abundant core prokaryotic taxa occurred among all varieties. Among these core taxa, we identified 49 and 108 heritable amplicon sequence variants, whose variations in relative abundances across the root endosphere and rhizosphere samples were significantly affected by wheat variety. The significant correlations between phylogenetic distance of wheat varieties and prokaryotic community dissimilarity were only observed in non-core and abundant subcommunities in the endosphere samples. Again, wheat yield was only significantly associated with root endosphere microbiota at the heading stage. Additionally, wheat yield could be predicted using the total abundance of 94 prokaryotic taxa as an indicator. Our results demonstrated that the prokaryotic communities in the root endosphere had higher correlations with wheat yield and quality than those in the rhizosphere; thus, managing root endosphere microbiota, especially core taxa, through agronomic practices and crop breeding, is important for promoting wheat yield and quality.

A Novel Ensemble Fault Diagnosis Model for Main Circulation Pumps of Converter Valves in VSC-HVDC Transmission Systems.

The intelligent fault diagnosis of main circulation pumps is crucial for ensuring their safe and stable operation. However, limited research has been conducted on this topic, and applying existing fault diagnosis methods designed for other equipment may not yield optimal results when directly used for main circulation pump fault diagnosis. To address this issue, we propose a novel ensemble fault diagnosis model for the main circulation pumps of converter valves in voltage source converter-based high voltage direct current transmission (VSG-HVDC) systems. The proposed model employs a set of base learners already able to achieve satisfying fault diagnosis performance and a weighting model based on deep reinforcement learning that synthesizes the outputs of these base learners and assigns different weights to obtain the final fault diagnosis results. The experimental results demonstrate that the proposed model outperforms alternative approaches, achieving an accuracy of 95.00% and an F1 score of 90.48%. Compared to the widely used long and short-term memory artificial neural network (LSTM), the proposed model exhibits improvements of 4.06% in accuracy and 7.85% in F1 score. Furthermore, it surpasses the latest existing ensemble model based on the improved sparrow algorithm, with enhancements of 1.56% in accuracy and 2.91% in F1 score. This work presents a data-driven tool with high accuracy for the fault diagnosis of main circulation pumps, which plays a critical role in maintaining the operational stability of VSG-HVDC systems and satisfying the unmanned requirements of offshore flexible platform cooling systems.

Design and Analysis of a Cardioid Flow Tube Valveless Piezoelectric Pump for Medical Applications.

Piezoelectric pumps play an important role in modern medical technology. To improve the flow rate of valveless piezoelectric pumps with flow tube structures and promote the miniaturization and integration of their designs, a cardioid flow tube valveless piezoelectric pump (CFTVPP) is proposed in this study. The symmetric dual-bend tube design of CFTVPP holds great potential in applications such as fluid mixing and heat dissipation systems. The structure and working principle of the CFTVPP are analyzed, and flow resistance and velocity equations are established. Furthermore, the flow characteristics of the cardioid flow tube (CFT) are investigated through computational fluid dynamics, and the output performance of valveless piezoelectric pumps with different bend radii is studied. Experimental results demonstrate that CFTVPP exhibits the pumping effect, with a maximum vibration amplitude of 182.5 µm (at 22 Hz, 100 V) and a maximum output flow rate of 5.69 mL/min (at 25 Hz, 100 V). The results indicate that a smaller bend radius of the converging bend leads to a higher output flow rate, while the performance of valveless piezoelectric pumps with different diverging bends shows insignificant differences. The CFTVPP offers advantages such as a high output flow rate, low cost, small size for easy integration, and ease of manufacturing.

Magnetic Field and Gravitational Waves from the First-Order Phase Transition.

We perform the three-dimensional lattice simulation of the magnetic field and gravitational wave productions from bubble collisions during the first-order electroweak phase transition. Except for the gravitational wave, the power-law spectrum of the magnetic field strength is numerically calculated for the first time, which is of a broken power-law spectrum: B_{ξ}∝f^{0.91} for the low-frequency region of ff_{⋆} in the thin-wall limit, with the peak frequency being f_{⋆}∼5 Hz at the phase transition temperature 100 GeV. When the hydrodynamics is taken into account, the generated magnetic field strength can reach B_{ξ}∼10^{-7} G at a correlation length ξ∼10^{-7} pc, which may seed the large scale magnetic fields. Our study shows that the measurements of cosmic magnetic field strength and gravitational waves are complementary to probe new physics admitting electroweak phase transition.

Exonuclease III-Powered Self-Propelled DNA Machine for Distinctly Amplified Detection of Nucleic Acid and Protein.

Herein, a new exonuclease III (Exo III)-powered self-propelled DNA machine was developed for the cascade multilevel signal amplification of nucleic acid and nucleic acid-related analytes. It could be easily and homogeneously operated with the use of an integral DNA hybrid probe as the recognition, amplification, and signaling element, and the Exo III cleavage as a driving force. The DNA hybrid probe was obtained by annealing two hairpin-like DNAs. The target recognition with the 3'-protruding domain of the DNA hybrid probe triggered Exo III cleavage, accompanied by target recycling and alternate generation of a large amount of target substitute and analogy. Simultaneously, the cascade bidirectional Exo III cleavage toward the DNA hybrid probe by the generated target substitute and analogy contributed for the exponential signal amplification toward target recognition event. It could be also extended for the application in protein detection with the thrombin as a protein example by introducing an additional hairpin-like aptamer switch. The proposed Exo III-powered self-propelled DNA amplification strategy showed a linear detection range for target DNA from 0.5 fM to 1 pM and for thrombin from 5 fM to 10 pM. The low detection limit toward target DNA and thrombin could reach about 0.1 fM and 5 fM, respectively, which were superior to most of reported methods. It also exhibited an excellent selectivity toward target detection. Therefore, the developed sensing system exhibits a new, simple and powerful means for amplified detection of nucleic acid and nucleic acid-related analytes, and may hold great potentials in bioanalysis, disease diagnosis and biomedicine.

Quantitative and Localization Fault Diagnosis Method of Rolling Bearing Based on Quantitative Mapping Model.

Aiming to solve the problem of accurate diagnosis of the size and location of rolling bearing faults, a novel quantitative and localization fault diagnosis method of the rolling bearing is proposed based on the quantitative mapping model (QMM). The fault size and location of the rolling bearing affect the impulse type and the modulation degree of the vibration signal, which subsequently changes the complexity and randomness of the time-domain distribution of the vibration signal. According to the relationship between the multiscale permutation entropy (MPE) of the vibration signal and rolling bearing fault size, an average MPE (A-MPE) index is proposed to establish linear and nonlinear QMMs through the regression function. The proper QMM is selected through the error rate of fault size prediction to achieve a quantitative fault diagnosis of the rolling bearing. Due to the mathematical characteristics of the QMM, the localization fault diagnosis is realized. The multiscale morphological filtering (MMF) method is also introduced to extract the time-domain geometric feature of the fault bearing vibration signal and to improve the QMM accuracy of the fault size prediction. The results show that the QMM has a great effect on the quantitative fault size prediction and localization diagnosis of the rolling bearing.

In Vivo and in Vitro Study on Drug-Drug Interaction of Lovastatin and Berberine from Pharmacokinetic and HepG2 Cell Metabolism Studies.

BACKGROUND: We assumed that the pharmacokinetics of lovastatin could be changed by the induction effect of berberine. METHODS: An UPLC-MS/MS method was developed and validated for the pharmacokinetics tudy of lovastatin to investigate the in vivo drug-drug interactions between lovastatin and berberine. SD male rats were random divided into lovastatin group and berberine induced prior to lovastatin group for the in vivo pharmacokinetic studies. Meanwhile HepG2 cells were induced by berberine for three days to study the metabolism of lovastatin. RESULTS: The AUC (p < 0.01) and Cmax (p < 0.01) could be significantly decreased in the berberine-induced group in vivo, and the metabolic activity of HepG2 cell ccould be increased by berberine induction in vitro. The metabolism parameters of lovastatin such as CL, Vmax and Km were increased after the induction of berberine. From the pharmacokinetic study of lovastatin induced with berberine, we obtained pharmacokinetic parameters which are compliance with the metabolic parameters of lovastatin in HepG2 cells with berberine induction in vitro. CONCLUSIONS: From the in vivo pharmacokinetics study and the HepG2 cell metabolism study in vitro, berberine could be an inducer for the metabolism of lovastatin according to our previous research on berberine induction effects on HepG2 cells, which may be relevant to the fact that berberine possesses induction effects through the CYP 450 3A4 enzyme.

Effects of Kaixinjieyu, a Chinese herbal medicine preparation, on neurovascular unit dysfunction in rats with vascular depression.

BACKGROUND: Kaixinjieyu (KJ), derived from Kaixin and Sini powder, is an effective Chinese herbal medicine preparation used in the treatment of vascular depression (VD). We hypothesize that broad antidepressant effect of KJ results from the improved neurovascular unit (NVU) function via neurogenesis, permeability of blood-brain barrier (BBB) and balance of the fibrinolytic system. METHODS: A VD model of rat was established by chronic unpredictable mild stress and separation after ligation of the bilateral common carotid arteries. The rats were treated with KJ and fluoxetine hydrochloride (FLU) for 21 days, respectively. The behavior and cerebral perfusion were investigated and then NVU functions including neurogenesis, permeability of BBB and balance of the fibrinolytic system were studied using a number of biomarkers and TUNEL assay. RESULTS: KJ significantly increased sucrose preference, moving distance, number of rearing and cortical blood flow. NVU functions measured by brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB) and tissue plasminogen activator (t-PA) proteins and mRNA, zona occludens protein-1 (ZO-1), occludin and claudin-5 proteins increased significantly, whereas, plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-2 (MMP-2) proteins, mRNA and apoptotic rates of neurons decreased significantly with treatment of KJ. FLU has a function similar to KJ in behavior, regulation of BDNF, TrkB, MMP-2, occludin and apoptotic rates of cells. CONCLUSIONS: KJ has function of reducing depression-like behavior and improving cerebral hypoperfusion, which might be mediated by the up-regulation of neurogenesis and tight junction of BBB, and balance of the fibrinolytic system. The results imply that KJ is better than FLU in improving cerebral hypoperfusion and the fibrinolytic system.

Novel antimony phosphates with enlarged birefringence induced by lone pair cations.

Phosphates, whose obvious disadvantage is the relatively small birefringence, can be overcome by the introduction of post-transition metal cations containing stereochemically active lone-pair electrons. In this paper, two new compounds were successfully explored in the A-Sb-P-O system, i.e. Cs2Sb3O(PO4)3 (CsSbPO) and (NH4)2Sb4O2(H2O)(PO4)2[PO3(OH)]2 (NH4SbPOH). Transmission spectra show that CsSbPO has a surprising transmission range with a UV cutoff edge of 213 nm. First-principles calculations show that both compounds have a wide band gap (5.02 eV for CsSbPO and 5.30 eV for NH4SbPOH) and enlarged birefringence (Δn = 0.034@1064 nm for CsSbPO and Δn = 0.045@1064 nm for NH4SbPOH). The results of real-space atom-cutting investigations show that the distorted [SbOx] polyhedra originating from the asymmetric lone pair electrons give the main contribution to the total birefringence and overcome the disadvantage of small birefringence of phosphates but maintain wide transition windows.

Cavitation is the determining mechanism for the atomization of high-viscosity liquid.

Piezoelectric atomization is becoming mainstream in the field of inhalation therapy due to its significant advantages. With the rapid development of high-viscosity gene therapy drugs, the demand for piezoelectric atomization devices is increasing. However, conventional piezoelectric atomizers with a single-dimensional energy supply are unable to provide the energy required to atomize high-viscosity liquids. To address this problem, our team has designed a flow tube internal cavitation atomizer (FTICA). This study focuses on dissecting the atomization mechanism of FTICA. In contrast to the widely supported capillary wave hypothesis, our study provides evidence in favor of the cavitation hypothesis, proving that cavitation is the key to atomizing high-viscosity liquids with FTICA. In order to prove that the cavitation is the key to atomizing in the structure of FTICA, the performance of atomization is experimented after changing the cavitation conditions by heating and stirring of the liquids.

Biological roles and functional mechanisms of arenavirus Z protein in viral replication.

Arenaviruses can cause severe hemorrhagic fever diseases in humans, with limited prophylactic or therapeutic measures. A small RING-domain viral protein Z has been shown to mediate the formation of virus-like particles and to inhibit viral RNA synthesis, although its biological roles in an infectious viral life cycle have not been directly addressed. By taking advantage of the available reverse genetics system for a model arenavirus, Pichinde virus (PICV), we provide the direct evidence for the essential biological roles of the Z protein's conserved residues, including the G2 myristylation site, the conserved C and H residues of RING domain, and the poorly characterized C-terminal L79 and P80 residues. Dicodon substitutions within the late (L) domain (PSAPPYEP) of the PICV Z protein, although producing viable mutant viruses, have significantly reduced virus growth, a finding suggestive of an important role for the intact L domain in viral replication. Further structure-function analyses of both PICV and Lassa fever virus Z proteins suggest that arenavirus Z proteins have similar molecular mechanisms in mediating their multiple functions, with some interesting variations, such as the role of the G2 residue in blocking viral RNA synthesis. In summary, our studies have characterized the biological roles of the Z protein in an infectious arenavirus system and have shed important light on the distinct functions of its domains in virus budding and viral RNA regulation, the knowledge of which may lead to the development of novel antiviral drugs.

Synthesis, structures and properties of two new selenite optical materials: K2Zn3Se4O12 and K4Zn3V4Se2O19.

Two new selenites, K2Zn3Se4O12 (compound 1) and K4Zn3V4Se2O19 (compound 2), have been successfully synthesized by solid-state reactions in vacuum tubes. Compound 1 consists of a three-dimensional (3D) framework with [SeO3] triangular pyramids and [ZnO4] tetrahedra in the monoclinic space group P21/c (No. 14). Compound 1's cut-off edge is below 344 nm, based on its UV-Vis-NIR diffuse reflectance studies, and theoretical calculations indicate a birefringence of around 0.043 at 1064 nm. The two-dimensional layer of compound 2, in contrast, is made up of [SeO3] triangular pyramids, [ZnO4] tetrahedra, and [V4O13] tetrahedra. It crystallizes in the monoclinic space group C2/c (No. 15). Its UV-Vis-NIR diffuse reflectance studies demonstrate that the compound's cut-off edge is lower than 330 nm.

Positive and Negative Contribution from Lead-Oxygen Groups and Halogen Atoms to Birefringence: A First Principles Investigation.

Oxyhalides, containing oxygen and halogen atoms and combining the advantages of oxides and halides in geometry and optical response, have great potential in optical materials. In this study, the electronic structures and the optical properties of the Pb3O2X2 (X = Cl, Br, I) compounds have been investigated using the first principles method. The results show that these compounds have birefringence at 0.076, 0.078, and 0.059 @ 1064 nm, respectively. And, the asymmetric stereochemical active lone pair electrons were found around lead atoms, which were confirmed by the projected density of states, the electronic localization functions, and the crystal orbitals. The contribution of atoms and polyhedra to birefringence was further evaluated using the Born effective charge. The results show that halogen atoms give negative contribution, and lead-oxygen polyhedra give positive contribution. The spin-orbit coupling effect is also investigated, and the downshift of the conduction band and variation in the valence band are found after relevant spin-orbit coupling (SOC), which leads to a reduction in the band gap and birefringence.

Improved Calculation Method of TAD for Intertrochanteric Fractures.

Purpose: To investigate the relative position of femur fixed screws using intramedullary systems for intertrochanteric fractures and to improve the accurate measurement method of the tip-to-apex distance (TAD) while providing a theoretical basis for the clinical treatment of such fractures. Methods: In the anteroposterior (AP) radiographs of the hip joint, the femoral neck axis through the femoral head geometry point was designated as the X-axis, while the line perpendicular to the X-axis passing through the femoral head geometry point was designated as the Y-axis. In the lateral radiographs of the hip joint, the line perpendicular to the X-axis passing through the femoral head geometry point was identified as the Z-axis. The head of the nail tip's location projected on the three axes was described as A AP, B in the AP radiographs; and A LAT, C in the lateral radiograph. The TAD was described as X AP and X LAT. The radius of the femoral head was D. All distance units were expressed in mm. Results: When the lateral projection angle was standardized, the A AP was equal to the A LAT, while the X AP 2=B 2+(D - A AP)2 and X LAT 2=C 2+(D - A LAT)2. When the lateral projection angle was not standardized, the value of C had no significant change; however, the (D - A LAT) value changed. Conclusions: The measurement value did not match the actual values of TAD when the lateral projection angle was not standardized, possibly leading to a misinterpretation during clinical work. The X LAT should be amended using the formula X LAT 2=C 2+(D - A AP)2.

Integrated organic and inorganic fertilization and reduced irrigation altered prokaryotic microbial community and diversity in different compartments of wheat root zone contributing to improved nitrogen uptake and wheat yield.

The effect of long-term water and integrated fertilization on prokaryotic microorganisms and their regulation for crop nutrient uptake remains unknown. Therefore, the impact of soil water and integrated fertilization after eight years on prokaryotic microbial communities in different compartments of root zone and their association with wheat nitrogen (N) absorption and yield were investigated. The results showed that compared with fertilization treatments (F), water regimes (W) more drastically modulated the prokaryotic microbial community structure and diversity in bulk soil, rhizosphere and endosphere. The increase of irrigation improved the prokaryotic diversity in the rhizosphere and endosphere while decreased the diversity in the bulk soil. Application of organic fertilizers significantly improved soil organic matter (SOM) and nutrient contents, increased rhizosphere and endophytic prokaryotic microbial diversity, and elevated the relative abundance of aerobic ammonia oxidation and nitrification-related functional microorganisms in rhizosphere and endosphere. Increasing irrigation elevated the relative abundance of functional microorganisms related to aerobic ammonia oxidation and nitrification in the rhizosphere and endosphere. Soil water content (SWC) and NH4+-N as well as NO3--N were key predictors of prokaryotic microbial community composition under W and F treatments, respectively. Appropriate application of irrigation and organic fertilizers increased the relative abundance of some beneficial bacteria such as Flavobacterium. Water and fertilization treatments regulated the prokaryotic microbial communities of bulk soil, rhizosphere and endosphere by altering SWC and SOM, and provided evidence for the modulation of prokaryotic microorganisms to promote nitrogen uptake and wheat yield under long-term irrigation and fertilization. Conclusively, the addition of organic manure (50 %) with inorganic fertilizers (50 %) and reduced amount of irrigation (pre-sowing and jointing-period irrigation) decreased the application amount of chemical fertilizers and water, while increased SOM and nutrient content, improved prokaryotic diversity, and changed prokaryotic microbial community structure in the wheat root zone, resulting in enhanced nutrient uptake and wheat yield.

Microwave-Assisted Coal-Derived Few-Layer Graphene as an Anode Material for Lithium-Ion Batteries.

A few-layer graphene (FLG) composite material was synthesized using a rich reservoir and low-cost coal under the microwave-assisted catalytic graphitization process. X-ray diffraction, Raman spectroscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy were used to evaluate the properties of the FLG sample. A well-developed microstructure and higher graphitization degree were achieved under microwave heating at 1300 °C using the S5% dual (Fe-Ni) catalyst for 20 min. In addition, the synthesized FLG sample encompassed the Raman spectrum 2D band at 2700 cm-1, which showed the existence of a few-layer graphene structure. The high-resolution TEM (transmission electron microscopy) image investigation of the S5% Fe-Ni sample confirmed that the fabricated FLG material consisted of two to seven graphitic layers, promoting the fast lithium-ion diffusion into the inner surface. The S5% Fe-Ni composite material delivered a high reversible capacity of 287.91 mAhg-1 at 0.1 C with a higher Coulombic efficiency of 99.9%. In contrast, the single catalyst of S10% Fe contained a reversible capacity of 260.13 mAhg-1 at 0.1 C with 97.96% Coulombic efficiency. Furthermore, the dual catalyst-loaded FLG sample demonstrated a high capacity-up to 95% of the initial reversible capacity retention-after 100 cycles. This study revealed the potential feasibility of producing FLG materials from bituminous coal used in a broad range as anode materials for lithium-ion batteries (LIBs).

Development of infectious clones for virulent and avirulent pichinde viruses: a model virus to study arenavirus-induced hemorrhagic fevers.

Several arenaviruses can cause hemorrhagic fever diseases (VHFs) in humans, the pathogenic mechanism of which is poorly understood due to their virulent nature and the lack of molecular clones. A safe, convenient, and economical small animal model of arenavirus hemorrhagic fever is based on guinea pigs infected by the arenavirus Pichinde (PICV). PICV does not cause disease in humans, but an adapted strain of PICV (P18) causes a disease in guinea pigs that mimics arenavirus hemorrhagic fever in humans in many aspects, while a low-passaged strain (P2) remains avirulent in infected animals. In order to identify the virulence determinants within the PICV genome, we developed the molecular clones for both the avirulent P2 and virulent P18 viruses. Recombinant viruses were generated by transfecting plasmids that contain the antigenomic L and S RNA segments of PICV under the control of the T7 promoter into BSRT7-5 cells, which constitutively express T7 RNA polymerase. By analyzing viral growth kinetics in vitro and virulence in vivo, we show that the recombinant viruses accurately recapitulate the replication and virulence natures of their respective parental viruses. Both parental and recombinant virulent viruses led to high levels of viremia and titers in different organs of the infected animals, whereas the avirulent viruses were effectively controlled and cleared by the hosts. These novel infectious clones for the PICV provide essential tools to identify the virulence factors that are responsible for the severe VHF-like disease in infected animals.