cTo evaluate vaccine efficacy in protecting against coxsackievirus A16 (CA16), which causes human hand, foot, and mouth disease (HFMD), we established the first neonatal mouse model. In this article, we report data concerning CA16-induced pathological changes, and we demonstrate that anti-CA16 antibody can protect mice against lethal challenge and that the neonatal mouse model could be used to evaluate vaccine efficacy. To establish a mouse model, a BJCA08/CA16 strain (at 260 50% lethal doses [LD 50 ]) was isolated from a patient and used to intracerebrally (i.c.) inoculate neonatal mice. The infection resulted in wasting, hind-limb paralysis, and even death. Pathological examination and immunohistochemistry (IHC) staining indicated that BJCA08 had a strong tropism to muscle and caused severe necrosis in skeletal and cardiac muscles. We then found that BJCA08 pretreated with goat anti-G10/CA16 serum could significantly lose its lethal effect in neonatal mice. When the anti-G10 serum was intraperitoneally (i.p.) injected into the neonatal mice and, within 1 h, the same mice were intracerebrally inoculated with BJCA08, there was significant passive immunization protection. In a separate experiment, female mice were immunized with formaldehyde-inactivated G10/CA16 and BJCA08/CA16 and then allowed to mate 1 h after the first immunization. We found that there was significant protection against BJCA08 for neonatal mice born to the immunized dams. These data demonstrated that anti-CA16 antibody may block virus invasion and protect mice against lethal challenge, and that the neonatal mouse model was a viable tool for evaluating vaccine efficacy. C oxsackievirus A16 (CA16) belongs to the Enterovirus genus of the Picornaviridae family and is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD) (4,16,19). CA16 was first isolated in 1951 (43). It is a single positive-stranded RNA virus and has an icosahedral symmetry structure. Its genome has approximately 7,410 nucleotides with one predominant serotype. Based on the VP4 nucleotide sequences, CA16 is classified into three distinct genetic lineages: A, B, and C. Before the 1990s, lineages A and B were the major epidemic strains in Asia (predominantly the B strain). After that, the CA16 gene gradually mutated to form lineage C, which replaced the B strain as the predominant epidemic strain (22).Epidemics of HFMD have been reported in England, Australia, Japan, Germany, Malaysia, Singapore, mainland China, and Taiwan (2,4,11,19,23,27,32,38,41,48). Recently, HFMD was highly epidemical in the west Pacific region, resulting in severe illness and fatalities (15, 23). The HFMD epidemics were mainly caused by CA16 and human enterovirus 71 (EV71), which circulated alternatively or together in the epidemic area (19,22,23,25,37). Because the most severe or fatal cases were caused by EV71, studies have mainly focused on EV71 but not CA16. However, in England, the largest HFMD outbreak (in 1994) was caused by CA16 (2). Similarly, in Taiwan the leading cau...

BackgroundPorcine circovirus type 2 (PCV2) is considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for the swine industry worldwide. The major genotypes, PCV2a and PCV2b, are highly prevalent in the pig population and are present worldwide. However, another newly emerging PCV2b genotype mutant, which has a mutation in its ORF2-encoded capsid protein, has been sporadically present in China, as well as in other countries. It is therefore important to determine the relative virulence of the newly emerging PCV2b genotype mutant, compared with the existing PCV2a and PCV2b genotypes, and to investigate whether the newly emerging mutant virus induces more severe illness.Methodology/Principal FindingsTwenty healthy, 30-day-old, commercial piglets served as controls or were challenged with PCV2a, PCV2b and the newly emerging mutant virus. A series of indexes representing different parameters were adopted to evaluate virulence, including clinical signs, serological detection, viral load and distribution, changes in immune cell subsets in the peripheral blood, and evaluation of pathological lesions. The newly emerging PCV2 mutant demonstrated more severe signs compatible with PMWS, characterized by wasting, coughing, dyspnea, diarrhea, rough hair-coat and depression. Moreover, the pathological lesions and viremia, as well as the viral loads in lymph nodes, tonsils and spleen, were significantly more severe (P<0.05) for piglets challenged with the newly emerging mutant compared with those in the groups challenged with PCV2a and PCV2b. In addition, a significantly lower average daily weight gain (P<0.05) was recorded in the group challenged with the newly emerging PCV2 mutant than in the groups challenged with the prevailing PCV2a and PCV2b.ConclusionsThis is believed to be the first report to confirm the enhanced virulence of the newly emerging PCV2 mutant in vivo.

All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium-or irondependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

BackgroundIncreasing rice (Oryza sativa L.) yield is a crucial challenge for modern agriculture. The ideal plant architecture is considered to be critical to enhance rice yield. Elite plant morphological traits should include compact plant type, short stature, few unproductive tillers, thick and sturdy stems and erect leaves. To reveal the genetic variations of important morphological traits, 523 germplasm accessions were genotyped using the Illumina custom-designed array containing 5,291 single nucleotide polymorphisms (SNPs) and phenotyped in two independent environments. Genome-wide association studies were performed to uncover the genotypic and phenotypic variations using a mixed linear model.ResultsIn total, 126 and 172 significant loci were identified and these loci explained an average of 34.45 % and 39.09 % of the phenotypic variance in two environments, respectively, and 16 of 298 (~5.37 %) loci were detected across the two environments. For the 16 loci, 423 candidate genes were predicted in a 200-kb region (±100 kb of the peak SNP). Expression-level analyses identified four candidate genes as the most promising regulators of tiller angle. Known (NAL1 and Rc) and new significant loci showed pleiotropy and gene linkage. In addition, a long genome region covering ~1.6 Mb on chromosome 11 was identified, which may be critical for rice leaf architecture because of a high association with flag leaf length and the ratio of flag leaf length and width. The pyramid effect of the elite alleles indicated that these significant loci could be beneficial for rice plant architecture improvements in the future. Finally, 37 elite varieties were chosen as breeding donors for further rice plant architectural modifications.ConclusionsThis study detected multiple novel loci and candidate genes related to rice morphological traits, and the work demonstrated that genome-wide association studies are powerful strategies for uncovering the genetic variations of complex traits and identifying candidate genes in rice, even though the linkage disequilibrium decayed slowly in self-pollinating species. Future research will focus on the biological validation of the candidate genes, and elite varieties will also be of interest in genome selection and breeding by design.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2245-2) contains supplementary material, which is available to authorized users.

Main conclusionTotally, 23 loci were detected, and 383 candidate genes were identified, and four of these candidate genes,Os01g0392100, Os04g0630000, Os01g0904700andOs07g0615000, were regarded as promising targets.Direct-seeding cultivation is becoming popular in rice (Oryza sativa L.)-planting countries because it is labor- and time-efficient. However, low seedling establishment and slow seedling emergence have restricted the application and popularity of the technique. Mesocotyl elongation and shoot length are two important traits that can enhance rice seedling emergence. A single nucleotide polymorphism (SNP) is a genome sequence variation caused by a single base within a population, and SNPs evenly distributed throughout the genomes of plant species. In this study, a genome-wide association study (GWAS), based on 4136 SNPs, was performed using a compressed mixed linear model that accounted for population structure and relative kinship to detect novel loci for the two traits. Totally, 23 loci were identified, including five loci located known QTLs region. For the mesocotyl elongation, 17 major loci were identified, explaining ~19.31 % of the phenotypic variation. For the shoot length, six major loci were detected, explaining ~39.79 % of the phenotypic variation. In total, 383 candidate genes were included in a 200-kb genomic region (±100 kb of each locus). Additionally, 32 SNPs were identified in 30 candidate genes. Relative expression level analyses indicated that four candidate genes containing SNP variations, Os01g0392100, Os04g0630000, Os01g0904700 and Os07g0615000, represented promising targets. Finally, eight elite accessions with long mesocotyl and shoot lengths were chosen as breeding donors for further rice direct-seeding variety modifications.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-015-2434-x) contains supplementary material, which is available to authorized users.

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BackgroundRice blast disease is one of the most serious and recurrent problems in rice-growing regions worldwide. Most resistance genes were identified by linkage mapping using genetic populations. We extensively examined 16 rice blast strains and a further genome-wide association study based on genotyping 0.8 million single nucleotide polymorphism variants across 366 diverse indica accessions.ResultsTotally, thirty associated loci were identified. The strongest signal (Chr11_6526998, P =1.17 × 10−17) was located within the gene Os11g0225100, one of the rice Pia-blast resistance gene. Another association signal (Chr11_30606558) was detected around the QTL Pif. Our study identified the gene Os11g0704100, a disease resistance protein containing nucleotide binding site-leucine rich repeat domain, as the main candidate gene of Pif. In order to explore the potential mechanism underlying the blast resistance, we further examined a locus in chromosome 12, which was associated with CH149 (P =7.53 × 10−15). The genes, Os12g0424700 and Os12g0427000, both described as kinase-like domain containing protein, were presumed to be required for the full function of this locus. Furthermore, we found some association on chromosome 3, in which it has not been reported any loci associated with rice blast resistance. In addition, we identified novel functional candidate genes, which might participate in the resistance regulation.ConclusionsThis work provides the basis of further study of the potential function of these candidate genes. A subset of true associations would be weakly associated with outcome in any given GWAS; therefore, large-scale replication is necessary to confirm our results. Future research will focus on validating the effects of these candidate genes and their functional variants using genetic transformation and transferred DNA insertion mutant screens, to verify that these genes engender resistance to blast disease in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0311-6) contains supplementary material, which is available to authorized users.

Biological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiological limitations. In this report, the entire K. pneumoniae nif gene cluster was reassembled as operon-based BioBrick parts in Escherichia coli. It provided ∼100% activity of native K. pneumoniae system. Based on the expression levels of these BioBrick parts, a T7 RNA polymerase–LacI expression system was used to replace the σ54-dependent promoters located upstream of nif operons. Expression patterns of nif operons were critical for the maximum activity of the recombinant system. By mimicking these expression levels with variable-strength T7-dependent promoters, ∼42% of the nitrogenase activity of the σ54-dependent nif system was achieved in E. coli. When the newly constructed T7-dependent nif system was challenged with different genetic and physiological conditions, it bypassed the original complex regulatory circuits, with minor physiological limitations. Therefore, we have successfully replaced the nif regulatory elements with a simple expression system that may provide the first step for further research of introducing nif genes into eukaryotic organelles, which has considerable potentials in agro-biotechnology.