Rescue of an Enterotropic Newcastle Disease Virus Strain ZM10 From Cloned cDNA and Stable Expressing an Inserted Foreign Gene

Background: Newcastle disease virus (NDV) strain ZM10, a typical enterotropic avirulent vaccine strain, has been widely used in in China for chickens against Newcastle disease. To elucidate its enterotropic mechanism and develop recombiant multivalent vaccines based on it, the reverse genetics system for NDV ZM10 is an indispensable platform.Results: A full-length cDNA clone of NDV ZM10 and three supporting plasmids were constructed using the ligation-independent cloning (LIC) method. Recombinant NDV rZM10 was successfully rescued after these plasmids were co-transfected into BHK-21 cells. Besides, the recombinant virus rZM10-RFP encoding the red fluorescent protein was generated by inserting the RFP gene into the full-length clone of NDV between the P and M genes. These rescued viruses were genetically and biologically identical to the parental strain and showed similar growth kinetics. Conclusion: The recovery system of NDV ZM10 strain was established, and can be used as a foundation for research on the enterotropic mechanism and development of multivalent vaccines against viral diseases of livestock and poultry.

Development of a Novel Avian Vaccine Vector Derived From the Emerging Fowl Adenovirus 4

Severe hepatitis-hydropericardium syndrome (HHS) associated with a novel viral genotype, fowl adenovirus 4 (FAdV-4), has emerged and widely spread in China since 2015, causing severe economic losses to the poultry industry. We previously reported that the hexon gene is responsible for pathogenicity and obtained a non-pathogenic hexon-replacement rHN20 strain; however, the lack of information about the non-essential regions for virus replication limits the development of a FAdV-4 vector. This study first established an enhanced green fluorescent protein (EGFP)-indicator virus based on the FAdV-4 reverse genetic technique, effective for batch operations in the virus genome. Based on this, 10 open reading frames (ORFs) at the left end and 13 ORFs at the right end of the novel FAdV-4 genome were deleted separately and identified as non-essential genes for viral replication, providing preliminary insertion sites for foreign genes. To further improve its feasibility as a vaccine vector, seven combinations of ORFs were successfully replaced with EGFP without affecting the immunogenicity of the vector backbone. Finally, a recombinant rHN20-vvIBDV-VP2 strain, expressing the VP2 protein of very virulent infectious bursa disease virus (vvIBDV), was rescued and showed complete protection against FAdV-4 and vvIBDV. Thus, the novel FAdV-4 vector could provide sufficient protection for HHS and efficient exogenous gene delivery. Overall, our findings systemically identified 23 non-essential ORFs for FAdV-4 replication and seven foreign gene insertion regions, providing valuable information for an in-depth understanding of the novel FAdV-4 pathogenesis and development of multivalent vaccines.

Targeted Deletion of the Wx Gene First Intron via CRISPR/Cas9 Significantly Increases Grain Amylose Content in Rice With a Wxb Allele, but Not in Rice With a Wxa Allele

Background: Rice Waxy (Wx) gene plays a major role in seed amylose synthesis, and consequently controls grain amylose content. The expression of Wx gene is highly regulated at both transcriptional and post-transcriptional levels. Particularly, the GT/TT poplymorphism at the 5` splicing site of its 1st intron greatly affects this intron’s splicing efficiency and defines two predominant Wx alleles, Wxa and Wxb. Wxa rice often has intermediate to high amylose content, whereas Wxb rice has low to intermediate amylose content. A previous study indicates that rice Wx 1st intron significantly enhances gene expression when it is inserted into the 5` UTR (untranslated region) of a foreign gene. By deleting Wx 1st intron with the CRISPR/Cas9 technology, we intended to create a totally noval Wx allele, and further to investigate how the intron removal affects Wx gene expression and rice grain amylose content.Results: CRISPR/Cas9-mediated targeted deletion of Wx 1st intron was performed on 4 rice inbreds, KY131(Wxb), X32(Wxb), X35(Wxa) and X55(Wxlv). Complete deletion of the 1st intron occurred in 8.6%-11.8% of the primary transformants of these 4 inbreds. Transgene-free, homozygous mutants were obtained. Their grain amylose content and Wx gene expression were analyzed. Compared to the amylose content of wild type plants, mutants’ amylose content was significantly increased from 13.0% to about 24% in KY131 and X32 which both carried the Wxb allele. However, no significant differenece in aylose content was observed between wild type plants and mutants of X35 and X55 which carried the Wxa and Wxlv allele, respectively. Results of Wx gene expression analysis on wild type plants and mutants showed a high consistence with their amylose content results. Mutants of KY131 and X32 accumulated much more steady mRNA transcripts than their wild type plants, while steady mRNA level remained somehow unchanged between wild type plants and mutants of X35 and X55. Grain quality including appearance quality and ECQ(eating and cooking quality) that are tightly linked to amylose content was also evalued on wild type plants and mutants, and data were presented and analyzed. Conclusions:This study presents a novel and fast strategy to increase amylose content for rice inbreds carrying a Wxb allele. Our data strongly suggest that rice Wx 1st intron regulates Wx gene expression mainly at the post-transcriptinal level, not as previously thought that it influences Wx gene transcription as well. In addition, removal of the first intron creates a completely noval Wx allele. Further studies on this new Wx allele would provide invaluable insights into the regulation of Wx gene expression, which will help researchers to engineer more new alleles that leads to the breeding of rice cultivars with better eating and cooking quality.

A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production

Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed.

Identification of four insertion sites for foreign genes in a pseudorabies virus vector

Background Pseudorabies virus (PRV) is a preferred vector for recombinant vaccine construction. Previously, we generated a TK&gE-deleted PRV (PRVΔTK&gE−AH02) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4 ~ 5 week-old PRV antibody negative piglets and provide rapid and 100 % protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRVTK&gE−AH02 may be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression. Results In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S(UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 h post infection (P < 0.05). Moreover, at 12 h post infection, cells infected with PRV-S(UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)ΔTK/gE (P = 0.097) and PRV-S(UL46-27)ΔTK/gE (P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRVΔTK&gE−AH02). Conclusions This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.

Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

Strategy for generation of replication–competent recombinant rotaviruses expressing multiple foreign genes

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

The Tn7-based genomic integration is dependent on an attTn7 box in the glms gene and is site-specific with monocopy in Ralstonia solanacearum species complex

The Tn7-based genomic integration system enables direct insertion of foreign gene elements into chromosome downstream of glms in many bacteria species. The glms gene is greatly conserved in Ralstonia solanacearum species complex (RSSC), while its downstream regions are mostly different in the RSSC. Here, we provided genetic evidence to validate that this Tn7-integration is dependent on a conserved 30-bp motif in the glms, called attTn7 box, and artificial attTn7 boxes elsewhere are competent for the Tn7-integration, which is further confirmed to be simultaneous at downstream of both original and artificial attTn7 boxes using the PCR. With the whole genome re-sequencing on 500 Tn7-colonies, the Tn7-integration was confirmed to be site- specific at 25-bp downstream of glms with monocopy as chromosome of the RSSC. Characteristic of the monocopy in chromosome enables the Tn7-based complementation to fully restore phenotypes of mutants to those of parent strains that is advantageous than those based on plasmids with low-copy numbers. The Tn7-based genomic integration system provides a generally applicable and versatile genetic tool for studies of complementation, pathogenesis, overexpression, and in-vivo promoter activity assays with monocopy in the RSSC.