Insertion of exogenous genes within the ORF1b coding region of porcine astrovirus.

Porcine astrovirus (PAstV) is a common cause of diarrhea in swine farms. The current understanding of the molecular virology and pathogenesis of PAstV is incomplete, especially due to the limited functional tools available. Here, ten sites in the open reading frame 1b (ORF1b) of the PAstV genome were determined to tolerate random 15 nt insertions based on the infectious full-length cDNA clones of PAstV using transposon-based insertion-mediated mutagenesis of three selected regions of the PAstV genome. Insertion of the commonly used Flag tag into seven of the ten insertion sites allowed the production of infectious viruses and allowed their recognition by specifically labeled monoclonal antibodies. Indirect immunofluorescence showed that the Flag-tagged ORF1b protein partially overlapped with the coat protein within the cytoplasm. An improved light-oxygen-voltage (iLOV) gene was also introduced into these seven sites, and only one viable recombinant virus that expressed the iLOV reporter gene at the B2 site was recovered. Biological analysis of the reporter viruses showed that these exhibited similar growth characteristics to the parental virus, but they produced fewer infectious virus particles and replicated at a slower rate. The recombinant viruses containing iLOV fused to ORF1b protein, which maintained their stability and displayed green fluorescence for up to three generations after passaging in cell culture. The porcine astroviruses (PAstVs) expressing iLOV were then used to assess the in vitro antiviral activities of mefloquine hydrochloride and ribavirin. Altogether, the recombinant PAstVs expressing iLOV can be used as a reporter virus tool for the screening of anti-PAstV drugs as well as the investigation of PAstV replication and the functional activities of proteins in living cells.

Surface Topography Prediction Model in Milling of Thin-Walled Parts Considering Machining Deformation.

With the continuous improvement of the performance of modern aerospace aircraft, the overall strength and lightweight control of aircraft has become a significant feature of modern aerospace parts. With the wide application of thin-walled parts, the requirements for dimensional accuracy and surface quality of workpieces are increasing. In this paper, a numerical model for predicting surface topography of thin-walled parts after elastic deformation is proposed. In view of the geometric characteristics in the cutting process, the cutting force model of thin-walled parts is established, and the meshing relationship between the tool and the workpiece is studied. In addition, the influence of workpiece deformation is considered based on the beam deformation model. Cutting force is calculated based on deformed cutting thickness, and the next cutting-meshing relationship is predicted. The model combines the radial deflection of the workpiece in the feed direction and the changing meshing relationship of the tool-workpiece to determine the three-dimensional topography of the workpiece. The error range between the experimental and the simulation results of surface roughness is 7.45-13.09%, so the simulation three-dimensional morphology has good similarity. The surface topography prediction model provides a fast solution for surface quality control in the thin-walled parts' milling process.

Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus.

A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.

Detection and Genetic Diversity of a Novel Water Buffalo Astrovirus Species Found in the Guangxi Province of China.

Astroviruses (AstVs) are major causative agents of gastroenteritis and have been detected worldwide. Little is known about the prevalence of neurotropic AstVs in Chinese water buffaloes, but a novel species which is associated with encephalitis and meningitis has recently been found. In this study, based on nested RT-PCR, rapid amplification of the 3'-cDNA end (3'-RACE) and next-generation sequencing (NGS), we examined the infection of AstVs in water buffaloes in the Guangxi Province of China. The results showed that the AstV infection was found in 40% (6/15) of the farms examined, and the prevalence of AstV in their feces was 11% (33/297). In addition, two near-full-length and two complete open reading frame 2 (ORF2) genes of AstVs from fecal sources were sequenced. Phylogenetic analysis of the ORF2 sequences indicated three lineages of BufAstVs, BufAstV lineage 1 was close related to the BoAstV, lineage 2 was related to the BufAstVs, and lineage 3 was classified as novel AstVs, which had a close relationship with the neurotropic/neurovirulent AstVs strains found in bovine, ovine, and musks. Moreover, genomic a recombination between the BufAstV and BoAstV strains was identified. This is a novel study reporting the genetic diversity of BufAstV infection in China especially found the similar neurotropic strains from fecal sources of water buffaloes, and it also provides details of the epidemiology, genetic recombination, and interspecies transmission of BoAstV and BufAstV in water buffaloes from the Guangxi Province of China.

Identification of a novel protein in porcine astrovirus that is important for virus replication.

Overlapping genes are common in some RNA viruses. It has been proposed that a potential overlapping gene is the ORFX, here termed ORF2b, which overlaps the ORF2 coding sequence in astroviruses. The aim of this study was to determine whether ORF2b is an overlapping gene that encodes a functional protein which is needed for viral replication. Sequence alignment showed that there was an ORF2b in a PAstV type 1 strain of astrovirus, PAstV1-GX1, which was embedded within the larger ORF2. The AUG codon for ORF2b is located 19 nucleotides downstream of the initiation site of ORF2 and contains 369 nucleotides and it codes for a predicted 122-amino-acid protein. A specific polyclonal antibody against the ORF2b protein was raised and used to demonstrate the expression of the new identified gene in virus-infected and pCAGGS-ORF2b-transfected cells. Analysis of purified virions revealed that the ORF2b protein was not incorporated into virus particles. Reverse genetics based on a PAstV type 1 infectious cDNA clone showed that the ORF2b protein was not essential but important for optimal virus infectivity. Knockout of the downstream potential stop codon candidate of ORF2b demonstrated that the C-terminus of the ORF2b protein can be extended by 170 amino acids, suggesting that the C-terminus of the newly identified ORF2b protein may be variable.