iCatch: a new strategy for capturing large DNA fragments using homing endonucleases.

Natural genetic materials contain many biosynthetic gene clusters encoding potentially valuable natural products, many of which can be used directly without codon optimization or other manipulations. With the development of synthetic biology, several DNA assembly standards have been proposed, conveniently facilitating the reuse of natural materials. Among these standards, the iBrick assembly standard was developed by our laboratory to manipulate large DNA fragments, employing two homing endonucleases. Considering the difficulty of cloning large iBrick parts using conventional endonuclease-mediated restriction and ligation methods, we herein present a new method, known as iCatch, which readily captures biosynthetic gene clusters. As the clusters cloned by iCatch have the prefix and suffix of the iBrick standard, they serve as new iBrick parts and are therefore conducive to further editing and assembly with the iBrick standard. iCatch employs the natural homologous recombination system to flank the region of interest with I-SceI and PI-PspI recognition sites, after which the genome is digested with I-SceI or PI-PspI and the fragments are then self-ligated to clone the target DNA fragments. We used this method to successfully capture the actinorhodin biosynthetic cluster from Streptomyces coelicolor and then heterologously expressed this cluster in a thermophilic Streptomyces strain. We propose that iCatch can be used for the cloning of DNA sequences that are dozens of kilobases in length, facilitating the heterologous expression of microbial natural products. Moreover, this cloning methodology can be a complementary tool for the iBrick standard, especially in applications requiring the manipulation of large DNA fragments.

Isolation and genome characterization of a novel duck Tembusu virus with a 74 nucleotide insertion in the 3' non-translated region.

During investigations into the outbreak of duck Tembusu virus (DTMUV) infection in 2011 in China, a DTMUV strain (DTMUV-AH2011) was isolated from the affected ducks. The length of the genome of the DTMUV-AH2011 strain was found to be 11,064 nucleotides and to possess 10,278 nucleotides of one open reading frame (ORF), flanked by 94 nucleotides of the 5' non-translated region (NTR) and 692 nucleotides of the 3' NTR. In comparison with five fully sequenced TMUV genomes, the genome of DTMUV-AH2011 had a 74 nucleotide insertion in the 3' NTR. Comparison of the DTMUV-AH2011 fully deduced amino acid sequences with those of other Tembusu virus strains reported recently in China showed they had a highly conserved polyprotein precursor, sharing 98.9% amino acid identities, at least. The overall divergences of amino acid substitutions were randomly distributed among viral proteins except for the protein NS4B, the protein NS4B was unchanged. Knowledge of the biological characters of DTMUV and the potential role of the insertion in the 3' NTR in RNA replication will be useful for further studies of the mechanisms of virus replication and pathogenesis.

Functional analysis of the interferon-stimulated response element of porcine circovirus type 2 and its role during viral replication in vitro and in vivo.

BACKGROUND: Porcine circovirus type 2 (PCV2) is associated with post-weaning multi-systemic wasting syndrome (PMWS) in young weaned pigs. Immune stimulation was found to activate the replication of PCV2 and exacerbate the clinical outcome of the infection. Proper amount of interferon-α (IFN-α) is able to enhance PCV2 infection and production in Porcine kidney-15 (PK-15) cells when administered after inoculation. METHODS: In the present study, luciferase reporter assays, construction of mutant viruses, Analysis the replication efficiency and the response to IFN-α treatment in PK-15 cells and animal experiments were carried out to analyze the function of interferon-stimulated response element (ISRE) of PCV2 and its role during viral replication in vitro and in vivo. RESULTS: A functional viral ISRE sequence, 5'-CTGAAAACGAAAGA-3', was identified in Rep gene promoter (Prep) of PCV2. PCV2 Prep is composed of two mini promoters, the proximal one span the sequence +1 to -106, containing an ISRE while the distal mini promoter is composed of three tandem GC box like sites locate at -85 to -194. It was demonstrated that viral ISRE is necessary for porcine IFN-α initiated luciferase expression enhancement and it plays an important role in affecting the replication efficiency of PCV2 in vivo and in vitro. CONCLUSIONS: These findings provide a theoretical basis for the Phenomenon of immunostimulation is able to enhance PCV2 infection, and improve the understanding of the complicated mechanisms involved in the host and pathogen interactions of PCV2.

Erratum: Author Correction: CRISPR-Cas12a-assisted nucleic acid detection.

[This corrects the article DOI: 10.1038/s41421-018-0028-z.].

[Secreted expression of Japanese encephalitis virus prME in Pichia pastoris and immunogenicity evaluation of the virus-like particles in mice].

The study was to express prME protein of Japanese encephalitis virus (JEV) in Pichia pastoris and then to evaluate the immunological properties of the recombinant protein in mice, so as to explore a new way for subunit vaccine development of JEV. The JEV prME gene was amplified by RT-PCR with genome RNA of JEV vaccine strain SA14-14-2 and subcloned into pPICZa-A vector, designated as pPICZα-prME. pPICZα-SprME was constructed same as pPICZα-prME besides with the additional 19 Aa signal peptides coding gene of the JEV cap protein C terminal. The linearized expression vector was integrated into the genome of Pichia pastoris X33 under the control of the alcohol oxidase (AOX1) promoter and induced with methanol during fermentation expression. The expression of JEV prME protein was identified by SDS-PAGE and Western blotting, and then it was purified by S-400 High Resolution HiPrep 16/60 Sephacry. The expressed products of Pichia pastoris were visualized by electron microscopy. In the immunization test, four groups of four-week old female mice were immunized subcutaneously with different doses purified JEV prME protein with complete Freund's adjuvant at a volumetric ratio of 1:1 and a control group was injected with sterile PBS. 10 µg/dose purified JEV prME protein mixing different doses nucleic acid adjuvant (Naa) was vaccinated in mice as the same mode. SDS-PAGE and Western blotting indicate that JEV prME was not cleaved between prM and E during secreted expression in Pichia pastoris. The purified recombinant prME was eluted in the first eluting peak which indicated that its molecular weight about 1×106 Da to 20×106 Da and may form a multimeric. Both the culture supernatant and the purified protein, examined by electron microscopy, we found to contain JEV virus like particles (VLPs) with diameters of 30-50 nm. The anti-JEV VLPs antibody titration reached peak at 3 wpi and still maintained in mice at 7 wpi inoculated with 10 µg and 15 µg prME. The strong antibody response was observed when the mice immunized with prME mixing nucleic acid adjuvant, which elicited high neutralizing antibody titer among 1:80 to 1:160. In conclusion, although JEV prME protein expressed in Pichia pastoris was not cleaved, which formed VLPs and showed efficient immunological properties in mice experiments.

Multiplex gene regulation by CRISPR-ddCpf1.

The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We demonstrated that ddCpf1 alone could be employed for gene repression in Escherichia coli, and the repression was more effective with CRISPR RNAs (crRNAs) specifically targeting to the template strand of its target genes, which was different from that of dCas9. When targeting the promoter region, both strands showed effective repression by the ddCpf1/crRNA complex. The whole-transcriptome RNA-seq technique was further employed to demonstrate the high specificity of ddCpf1-mediated repression. Besides, we proved that the remaining RNase activity in ddCpf1 was capable of processing a precursor CRISPR array to simply generate multiple mature crRNAs in vivo, facilitating multiplex gene regulation. With the employment of this multiplex gene regulation strategy, we also showed how to quickly screen a library of candidate targets, that is, the two-component systems in E. coli. Therefore, based on our findings here, the CRISPR-ddCpf1 system may be further developed and widely applied in both biological research and clinical studies.

MicroRNA transcriptome profiling of mice brains infected with Japanese encephalitis virus by RNA sequencing.

Japanese encephalitis (JE) is a mosquito borne viral disease, caused by Japanese encephalitis virus (JEV) infection producing severe neuroinflammation in the central nervous system (CNS) with the associated disruption of the blood brain barrier. MicroRNAs (miRNAs) are a family of 21-24 nt small non-coding RNAs that play important post-transcriptional regulatory roles in gene expression and have critical roles in virus pathogenesis. We examined the potential roles of miRNAs in JEV-infected suckling mice brains and found that JEV infection changed miRNA expression profiles when the suckling mice began showing nervous symptoms. A total of 1062 known and 71 novel miRNAs were detected in JEV-infected group, accompanied with 1088 known and 75 novel miRNAs in mock controls. Among these miRNAs, one novel and 25 known miRNAs were significantly differentially expressed, including 18 up-regulated and 8 down-regulated miRNAs which were further confirmed by real-time PCR. Gene ontology (GO) and signaling pathway analysis of the predicted target mRNAs of the modulated miRNAs showed that they are correlated with the regulation of apoptosis, neuron differentiation, antiviral immunity and infiltration of mouse brain, and the validated targets of 12 differentially expressed miRNAs were enriched for the regulation of cell programmed death, proliferation, transcription, muscle organ development, erythrocyte differentiation, gene expression, plasma membrane and protein domain specific binding. KEGG analysis further reveals that the validated target genes were involved in the Pathways in cancer, Neurotrophin signaling pathway, Toll like receptor signaling pathway, Endometrial cancer and Jak-STAT signaling pathway. We constructed the interaction networks of miRNAs and their target genes according to GO terms and KEGG pathways and the expression levels of several target genes were examined. Our data provides a valuable basis for further studies on the regulatory roles of miRNAs in JE pathogenesis.

The A66G back mutation in NS2A of JEV SA14-14-2 strain contributes to production of NS1' protein and the secreted NS1' can be used for diagnostic biomarker for virulent virus infection.

Japanese encephalitis virus (JEV) is the most common cause of the prevalent encephalitis in Asia-Pacific region and poses a serious risk to public health. Here, we developed a reliable reverse genetics system based on the JEV SA14-14-2 strain to further explore the mechanism for the synthesis of NS1' protein and to investigate the function of NS1' protein during virus infection. NS1' is an additional form of NS1 protein with 52 amino acid carboxy-terminal extension and is expressed by the members of the Japanese encephalitis (JE) serogroup due to the translation frameshift. A66G substitution in NS2A gene of JEV SA14-14-2 strain contributed to recover the GC-rich pseudoknot and resulted in the formation of the NS1'. The NS1' protein has no significant effect on the virus replication properties in BHK-21 cells. Animal experiments demonstrated that the NS1' protein had a rather minor effect on neurovirulence of JEV SA14-14-2 strain. But the NS1'-expressing virus (rA66G) could induce a higher humoral immune response than the NS1'-non-expressing virus (rSA14-14-2). NS1' protein can be detected in the serum of JEV rA66G infected animal and in the cultural media of that infected mammalian cells. Interesting, only the dimer of NS1' can be detected in the cultural media of the infected BHK-21 cells and the amount of the secreted NS1' was in agreement with that of the secreted virion. In comparison with the live-attenuated JE vaccine strain which is incapable of formation of NS1', most of the virulent JEV strains produce the NS1' protein. And the secreted NS1' may serve as an early surrogate biomarker for viremia to distinguish the field infection from the vaccine inoculation. In total, in the present study, we identified the nt 66 in the viral NS2A gene as one of the critical site for the -1 programmed ribosomal frameshift to produce the NS1' protein and demonstrated the secreted NS1' could be used for diagnostic biomarker during JEV infection.

Integration analysis of miRNA and mRNA expression profiles in swine testis cells infected with Japanese encephalitis virus.

To elucidate the role of microRNAs (miRNA) in the regulation of gene expression in Japanese encephalitis virus (JEV) infected swine testis (ST) cells, we analyzed miRNA and mRNA expression profiles of JEV infected ST cells by high-throughput sequencing technology as compared to uninfected controls. The results showed that 104 known miRNAs and 9 new miRNA candidates were differentially expressed in ST cells after JEV infection. We identified 396 differentially expressed mRNAs. Bioinformatics analysis identified 435 known miRNA-mRNA interaction pairs and 94 novel miRNA-mRNA interaction pairs involving miRNAs inversely correlated with the expression of their predicted target mRNAs. The known miRNAs inversely correlated with their target genes were involved in the biological processes of immunity, cytokine production, inflammation, and apoptosis. Selected miRNA-mRNA interactions were validated by luciferase reporter assay. Overall, our findings indicate that miRNAs may play critical roles in the pathogenesis of JEV infection.