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Yellow fever virus (YFV), the prototype of the Flavivirus genus, can cause devastating yellow fever disease. Although it is preventable by vaccination, there are still tens of thousands of yellow fever cases per year, and no approved antiviral medicine is available.

The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.

Objective: This study aimed to investigate the feasibility of repairing rabbit radius bone defects with simvastatin compound biological bone. Methods: Simvastatin biological bone material was prepared, and osteoblasts were cultured. A total of 42 New Zealand white rabbits were randomly divided into four groups, and a bone defect with a length of 15 mm was created at the middle part of the radial shaft of both limbs in each rabbit, thereby establishing a bone defect model. The grafts in group A were biological bones of osteoblasts combined with simvastatin; the grafts in group B were biological bones of simvastatin; the grafts in group C were biological compound bones of osteoblasts; and the grafts in group D were simple biological bones. In each group, four animals were randomly sacrificed at the sixth and twelfth week after surgery, and specimens were collected for gross observation, X-ray examination, histological observation, and biomechanical testing. In each group, two animals were randomly sacrificed at the twelfth week after surgery; a three-point bending test was performed using a biomechanical testing machine, and the results were compared with those of a normal radius. Results: The X-ray and histological examinations at 6 and 12 weeks after surgery revealed that the osteogenesis ability of the simvastatin biological bone and osteoblast-simvastatin biological bone was better than that of the osteoblast biological bone and simple biological bone, which was superior in group A and group B to group C and group D. The results of the biomechanical examination revealed that the maximum stress of the normal radius was significantly higher than that of the experimental groups. Among the experimental groups, the difference between group A and group B was not statistically significant, and the maximum stress was higher in groups A and B than in groups C and D. Conclusion: Simvastatin biological bone material can promote the repair of rabbit radius defects and increase the quality of bone healing.

In this manuscript, we consider a coupled AB system, which describes the baroclinic instability processes in the geophysical flows. Darboux-dressing transformation is used to derive the bright-dark soliton, breather and semirational rogue wave solutions for such a system. We observe that type of the solutions is relate to the spectral parameter $$\lambda $$ , amplitude $$a_{1}$$ and wave number q. Elastic collision between dark or bright solitons, propagations of the bright or dark breathers and rogue waves coexist with two dark or bright solitons are respectively illustrated in figures. The results about those localized wave phenomena are expected to have potential applications.

Full endoscopic transforaminal lumbar interbody fusion has been used widely in the field of minimally invasive spine surgery in recent years. This paper briefly introduces the development history, technical points, indications, curative effects and complications. Authors believe that the full endoscopic transforaminal lumbar interbody fusion has the same clinical effects as traditional surgery, and can effectively reduce tissue damage and intraoperative bleeding, reduce the incidence of postoperative low back pain, shorten the time to get out of bed, and reduce the average hospitalization time. However, it is still necessary to improve the long-term follow up in order to further evaluate the effectiveness and safety of the procedure.

Yellow fever virus (YFV) is a re-emerging flavivirus, which can lead to severe clinical manifestations and high mortality, with no specific antiviral therapies available. The live-attenuated yellow fever vaccine 17D (YF17D) has been widely used for over eighty years. However, the emergence of yellow fever vaccine-associated viscerotropic disease (YFL-AVD) and yellow fever vaccine-associated neurotropic disease (YFL-AND) raised non-negligible concerns. Additionally, the attenuation mechanism of YF17D is still unclear. Thus, the development of convenient models is crucial to understand the mechanisms behind YF17D attenuation and its adverse effects. In this work, we generated a reporter YF17D expressing nano-luciferase (NLuc). In vitro and in vivo characterization demonstrated that the NLuc-YF17D shared similar biological properties with its parental strain and the NLuc activity can reflect viral infectivity reliably. Combined with in vivo bioluminescence imaging, a series of mice models of YF17D infection was established, which will be useful for the evaluation of antiviral medicines and novel vaccine candidates. Especially, we demonstrated that intraperitoneally (i.p.) infection of NLuc-YF17D in type I interferon receptor-deficient mice A129 resulted in outcomes resembling YEL-AVD and YEL-AND, evidenced by viral replication in multiple organs and invasion of the central neuronal system. Finally, in vitro and in vivo assays based on this reporter virus were established to evaluate the antiviral activities of validated antiviral agents. In conclusion, the bioluminescent reporter virus described herein provides a powerful platform to study YF17D attenuation and vaccine-associated diseases as well as to develop novel countermeasures against YFV.

Yellow fever virus (YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans. Despite the availability of an effective vaccine, little is known about the replication mechanism of YFV, and there are still no available specific anti-YFV medicines. Herein, by introducing the Renilla luciferase gene (Rluc) into an infectious clone of YFV vaccine strain 17D, we generated a recombinant virus 17D-Rluc.2A via reverse genetics approaches. The 17D-Rluc.2A had similar plaque morphology and comparable in vitro growth characteristics with its parental strain. Importantly, the reporter luciferase was efficiently expressed in 17D-Rluc.2A-infected mammalian and mosquito cells, and there was a good linear correlation between intracellular luciferase expression and extracellular infectious virion reproduction. Furthermore, by a combination of the 17D-Rluc.2A reporter virus and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) technology, the conserved 5′-SLA element was shown to be essential for YFV replication, highlighting the capability of 17D-Rluc.2A in the investigation of YFV replication. At last, we demonstrated that two compounds with distinct anti-viral mechanisms can effectively inhibit the viral propagation in 17D-Rluc.2A-infected cells, demonstrating its potential application in the evaluation of anti-viral medicines. Taken together, the 17D-Rluc.2A serves as a useful tool for the study of YFV replication and anti-YFV medicine development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12250-021-00428-1.

With the popularization of intelligent mobile terminals, the emerging high-speed data exchange services make MIMO become an important development direction, which poses new challenges to the planning and optimization of communication networks. In order to support the design and optimization of future MIMO communication system suitable for complex indoor environment, the deterministic channel model based on ray tracing method is used to calculate the impulse response of the receiving antenna of MIMO system, which provides a reliable support for the channel capacity estimation in this scenario. The simulation results show that the channel capacity of the MIMO system increases with the increase of the antenna density, the antenna array aperture and the SNR, or decreases with the decrease of the spatial correlation between multipaths.

Dengue virus (DENV) infection can lead to a complex spectrum of clinical outcomes, ranging from asymptomatic infection to life-threatening severe dengue. The reasons for thus drastically varying manifestations of the disease remain an enigma. Herein, we reported an original discovery of the synergistic effect between preexisting Epstein–Barr virus (EBV) infection and DENV superinfection in vitro and of a strong correlation of these two viruses in the clinical samples from dengue patients. We showed that (I) DENV-2 infection of an EBV-positive cell line (EBV + Akata cell) reactivated EBV, and it could be blocked by wortmannin treatment. (II) Examination of human peripheral blood mononuclear cell (PBMC) samples from dengue patients revealed significantly elevated cell-associated EBV DNA copy number at the time of hospitalization vs. at the time of disease recovery in most individuals. (III) EBV infection promoted DENV propagation in both EBV-hosting B cells and indirectly in THP-1 cells, supported by the following evidence: (A) EBV + Akata cells were more permissive to DENV-2 infection compared with Akata cells harboring no EBV virus (EBV- Akata cells). (B) Low-molecular weight fraction secreted from EBV + Akata cells could enhance DENV-2 propagation in monocytic THP-1 cells. (C) While reactivation of EBV in EBV + Akata cells further increased DENV-2 yield from this cell line, pharmacological inhibition of EBV replication by acyclovir had the opposite effect. To our knowledge, this is the first investigation demonstrating a positive correlation between EBV and DENV in vitro and in human biospecimens.

On the basis of the propagation characteristics of electromagnetic wave attenuation, reflection, and diffraction, a imaging algorithm with an envelope of ellipses is proposed to obtain the image of the building geometry structure. The ellipse model regarding a target is established by analyzing the geometry relationship between the target and the coordinates of transceiver. With this model, it can get better imaging results using the echo delay.

In order to find the best wireless communication technology scheme for the wireless channel environment between ships on the sea surface, this paper presents a communication model for multipath between ships on the surface of the sea based on the ray tracing algorithm. The multipath model presented herein covers the direct and specular reflection and the diffuse components. In this paper, large-scale fading analyses on wireless channel of ship-to-ship communication are presented.

With the popularization of wireless networks, wireless positioning technology services have met people's positioning needs. How to reduce the cost and improve accuracy of positioning technology is very important. In complex urban environments, network-centric-warfare requires large-scale, portable and inexpensive devices for situational awareness of non-cooperative target sources. In this paper, a fingerprint location algorithm based on KNN classification algorithm is designed and validated. Ray-tracing based channel loss model is adopted in this algorithm for collecting fingerprint database instead of manual collection. Finally, the accuracy of the algorithm is verified by experiment data. The results show that the fingerprint database built with deterministic loss propagation model has a high positioning accuracy.

Zika virus induces the formation of vesicles from ER membrane that form viral replication factories in the ER lumen. Ci et al. show that the Zika NS1 protein plays a key role in this remodeling of the ER as the insertion of the hydrophobic regions of NS1 into the inner leaflet of the ER membrane creates the compartments essential for viral replication., Zika virus (ZIKV), a recently emerged member of the flavivirus family, forms replication compartments at the ER during its lifecycle. The proteins that are responsible for the biogenesis of replication compartments are not well defined. Here, we show that Zika nonstructural protein 1 (NS1)–induced ER remodeling is essential for viral replication. NS1 expressed in the ER lumen induced ER perinuclear aggregation with an ultrastructure resembling that of the replication compartment. Data from model membrane system indicated that the membrane-binding and membrane-remodeling properties of NS1 depend on its hydrophobic insertion into the membrane. These findings demonstrate that NS1 plays a crucial role in flavivirus replication compartment formation by remodeling the ER structure., Graphical Abstract

The genus Flavivirus is a group of single-stranded, positive-sense RNA viruses that includes numerous human pathogens with global impact, such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Zika virus (ZIKV). The approximately 11-kilobase genome is flanked by highly structured untranslated regions (UTRs), which contain various cis-acting RNA elements with unique structures and functions. Moreover, local RNA elements circularize the genome non-covalently through long-range interactions. Interestingly, many flavivirus cis-acting RNA elements contain group-specific motifs or are specific for the given phylogenetic groups, suggesting their potential association with flavivirus evolution and diversification. In this review, we summarize recent advances about the structure and function of cis-acting RNA elements in flavivirus genomes and highlight the potential implications for flavivirus evolution. Finally, the scientific questions remained to be answered in the field are also discussed.

Most mosquito‐borne flaviviruses, including Zika virus (ZIKV), Dengue virus (DENV), and West Nile virus (WNV), produce long non‐coding subgenomic RNAs (sfRNAs) in infected cells that link to pathogenicity and immune evasion. Until now, the structural characterization of these lncRNAs remains limited. Here, we studied the 3D structures of individual and combined subdomains of sfRNAs, and visualized the accessible 3D conformational spaces of complete sfRNAs from DENV2, ZIKV, and WNV by small angle X‐ray scattering (SAXS) and computational modeling. The individual xrRNA1s and xrRNA2s adopt similar structures in solution as the crystal structure of ZIKV xrRNA1, and all xrRNA1‐2s form compact structures with reduced flexibility. While the DB12 of DENV2 is extended, the DB12s of ZIKV and WNV are compact due to the formation of intertwined double pseudoknots. All 3′ stem‐loops (3′SLs) share similar rod‐like structures. Complete sfRNAs are extended and sample a large conformational space in solution. Our work not only provides structural insight into the function of flavivirus sfRNAs, but also highlights strategies of visualizing other lncRNAs in solution by SAXS and computational methods.

Zika virus (ZIKV) has evolved from an overlooked mosquito-borne flavivirus into a global health threat due to its astonishing causal link to microcephaly and other disorders. ZIKV has been shown to infect neuronal progenitor cells of the fetal mouse brain, which is comparable to the first-trimester human fetal brain, and result in microcephaly. However, whether there are different effects between the contemporary ZIKV strain and its ancestral strain in the neonatal mouse brain, which is comparable with the second-trimester human fetal brain, is unclear. Here we adopted a mouse model which enables us to study the postnatal effect of ZIKV infection. We show that even 100 pfu of ZIKV can replicate and infect neurons and oligodendrocytes in most parts of the brain. Compared with the ancestral strain from Cambodia (CAM/2010), infection of the ZIKV strain from Venezuela (VEN/2016) leads to much more severe microcephaly, accompanied by more neuronal cell death, abolishment of oligodendrocyte development, and a more dramatic immune response. The serious brain damage caused by VEN/2016 infection would be helpful to elucidate why the American strain resulted in severe neurovirulence in infants and will provide clinical guidance for the diagnosis and treatment of infection by different ZIKV strains., Highlights • The infection of an American strain of ZIKV leads to more severe microcephaly than the ancestral Asian strain. • American strain infects more cells, and induces more dramatic immune response and cell death than ancestral Asian strain. World attention has been drawn to a global Zika virus (ZIKV) outbreak due to its unexpected causal link to congenital brain abnormalities, especially microcephaly. Infection of pregnant women with the American Zika strain, but not the ancestral Asian strain, can result in microcephaly in infants. However, the phenotypic difference between the contemporary American strain and ancestral Asian strain of ZIKV is still unclear. We employed the ZIKV infection model of a neonatal mouse brain to compare the difference between these two strains. We find that infection by the American strain leads to more severe microcephaly than the ancestral Asian strain.

In the paper, for HH polarization the composite scattering from 2-D perfectly electric conducting (PEC) object situated above 1-D dielectric sea surface with large windspeed is investigated by an efficient hybrid method combining integral equation method with physical optics (IEM–PO). In the hybrid method, IEM is applied to study the direct scattering of underlying sea surface, whereas PO is employed to deal with the direct scattering of object. The mutual couplings between sea surface and object are also solved by PO method based on the equivalence principle and multi-path scattering strategy. In numerical simulations, the bistatic normalized radar cross-section (NRCS) of composite model are calculated by the proposed method and are validated by numerical experiments for different incident angles and windspeeds. The results show the hybrid IEM-PO method has a good accuracy and can greatly reduce the computational time and memory requirement.

The rapid spread and potential link with birth defects have made Zika virus (ZIKV) a global public health problem. The virus was discovered 70years ago, yet the knowledge about its genomic structure and the genetic variations associated with current ZIKV explosive epidemics remains not fully understood. In this review, the genome organization, especially conserved terminal structures of ZIKV genome were characterized and compared with other mosquito-borne flaviviruses. It is suggested that major viral proteins of ZIKV share high structural and functional similarity with other known flaviviruses as shown by sequence comparison and prediction of functional motifs in viral proteins. Phylogenetic analysis demonstrated that all ZIKV strains circulating in the America form a unique clade within the Asian lineage. Furthermore, we identified a series of conserved amino acid residues that differentiate the Asian strains including the current circulating American strains from the ancient African strains. Overall, our findings provide an overview of ZIKV genome characterization and evolutionary dynamics in the Americas and point out critical clues for future virological and epidemiological studies.

The study of electromagnetic wave propagation in complex environment has always been a hot spot. In this paper, firstly, the propagation mechanism of electromagnetic wave in medium was studied. Secondly, based on Snell’s law of electromagnetic wave, the propagation path of electromagnetic wave through a single simple building was recorded, and the electric field power coverage in the shadow area of the building was obtained through the propagation path, the simulation results are analyzed.

In order to get the radio propagation path through the atmosphere, a common ray tracing model is introduced and the acquisition methods of relevant parameters are given according to the actual situation in China. This calculation method can quickly simulate the electromagnetic wave propagation paths. The algorithm has the characteristics of high practicability and accuracy for calculating ray path. In this paper, a case has been studied for illustrating the advantages of using the ray tracing algorithm mentioned above.

In order to analyze coverage prediction of irregular terrain using ray tracing model, some form of accelerating must be performed to improve computational efficiency. A new image source tree structure is proposed to improving the efficiency of coverage prediction. This paper describes an accelerating algorithm that has proven effective in working with large terrain data files which extracted from SRTM Digital Elevation Model (DEM) database. On the basis of triangular subdivision of DEM data, a lot of coverage predictions are illustrated to analyze the field distribution and channel characteristic using ray-tracing method.

Zika virus (ZIKV) was once considered an obscure member of the large and diverse family of mosquito-borne flaviviruses, and human infections with ZIKV were thought to be sporadic, with mild and self-limiting symptoms. The large-scale ZIKV epidemics in the Americas and the unexpected uncovering of a link to congenital birth defects escalated ZIKV infections to the status of a global public health emergency. Recent studies that combined reverse genetics with modelling in multiple systems have provided evidence that ZIKV has acquired additional amino acid substitutions at the same time as congenital Zika syndrome and other birth defects were detected. In this Progress article, we summarize the evolution of ZIKV during its spread from Asia to the Americas and discuss potential links to pathogenesis.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs. In vivo probing of RNA structures with COMRADES yields insight into RNA folding of the ZIKA virus genome and its interaction with host RNAs.

Background/Aims: The pathphysiological properties of N-Methyl -N -nitrosourea (MNU) induced photoreceptor degeneration are similar to the hereditary retinitis pigmentosa (RP). The present study sought to explore the beneficial effects of the peonidin, a common aglycone form of anthocyanin, on the MNU induced photoreceptor degeneration via topographic measurements. Methods: The MNU administrated mouse received peonidin or vehicle injections, and then they were examined by electroretinography (ERG), multi electrode array (MEA), histological and immunohistochemistry studies. Results: The protective effects of peonidin on the MNU administrated retinas were systematically verified and quantified by topographic measures. The peonidin treatment could protect the photoreceptor against the MNU toxicity both functionally and morphologicaly. The most sensitive zone to peonidin therapy was sorted out, indicating that different rescuing kinetics existed between the retinal hemispheres and retinal quadrants. Moreover, the hyperactive spontaneous firing response and the debilitated light induced response in MNU administrated retinas could be partially reversed by peonidin treatment. To our knowledge, this was the first study to explore the pharmacological effects of peonidin on the electrophysiological properties of inner visual signal pathways. Conclusion: The peonidin could ameliorate the MNU induced photoreceptors degeneration and rectify the abnormities in the inner visual signal pathways. Future refinements of the knowledge cast insights into the discovery of a novel treatment for human RP.

The environmental information of the indoor scene has great influence on the prediction of the indoor visible light communication channel and the optimal layout of the light source. This article mainly introduces a modeling method for predicting the optical power distribution of indoor visible light communication when a simple furniture is arranged in the room. The method is based on the Lambertian model, which meshes the surface of a wall surface or an indoor object to obtain a small surface element. The microelement is used as a receiver or a secondary light source, and the optical power distribution of indoor visible light communication is simulated and analyzed. It provides a feasible modeling method for indoor visible light communication channel prediction and optimal layout of light sources.

Two-and-one-half dimensional ray tracing models, in combination with the uniform theory of diffraction and geometrical optics, are widely employed for propagation prediction in urban microcellular environments because of their high efficiency and reliable prediction accuracy. In this study, an improved ray tracing algorithm based on the “orientation face set” concept and on the improved 2D polar sweep algorithm is used. The aim of this paper is mainly to analyze the effects of different propagation mechanisms on ray-tracing-based coverage predictions in urban microcellular environments. In addition, the effects associated with the coverage prediction are visually represented.

Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis -acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5′-SLA promoter and 5′-3′ cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses. IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis -acting replication elements (5′-SLA and 5′-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.

Japanese encephalitis remains the leading cause of viral encephalitis in children in Asia and is expanding its geographical range to larger areas in Asia and Australasia. Five genotypes of Japanese encephalitis virus (JEV) co-circulate in the geographically affected areas. In particular, the emergence of genotype I (GI) JEV has displaced genotype III (GIII) as the dominant circulating genotype in many Asian regions. However, all approved vaccine products are derived from GIII strains. In the present study, bioinformatic analysis revealed that GI and GIII JEV strains shared two distinct amino acid residues within the envelope (E) protein (E222 and E327). By using reverse genetics approaches, A222S and S327T mutations were demonstrated to decrease live-attenuated vaccine (LAV) SA14-14-2-induced neutralizing antibodies in humans, without altering viral replication. A222S or S327T mutations were then rationally engineered into the infectious clone of SA14-14-2, and the resulting mutant strains retained the same genetic stability and attenuation characteristics as the parent strain. More importantly, immunization of mice with LAV-A222S or LAV-S327T elicited increased neutralizing antibodies against GI strains. Together, these results demonstrated that E222 and E327 are potential genotype-related neutralization determinants and are critical in determining the protective efficacy of live Japanese encephalitis vaccine SA14-14-2 against circulating GI strains. Our findings will aid in the rational design of the next generation of Japanese encephalitis LAVs capable of providing broad protection against all JEV strains belonging to different genotypes.

Development of agricultural biotechnology requires rapid and convenient methods for crop plant genotyping. Real-time PCR is sensitive and reliable, and has been a routine technique in plant research. However, its application is limited by the cumbersome DNAtemplate preparation procedures. We tested three PCRmaster mixes for direct amplification of crude seed DNA extracts without extensive purification. One mixhad higher resistance to plant-derived PCR inhibitors and was shown to be applicable to various important crop plants. Furthermore, this method is capable of detecting single-copy genes from 2 mg pieces of seeds repetitively. Meanwhile, melting curve analysis could detect amplicons directly without electrophoresis manipulations. Taken together, this direct real-time PCR method provides a rapid and convenient toolfor seed genotypic screening in crop plants.