| The influenza virus reverse genetics system can perform the viral genome in vitro, and facilitates to the study of influenza virus. The minimal replication unit of influenza virus is composed of ribonucleoprotein complex(RNP). In the influenza virus reverse genetics system, the(-)v RNA generated by RNA polymerase I(Pol I) promoter is recognized by the PB1,PB2,PA and NP protein. The(-)v RNA is then transcribed into m RNAs and(+)c RNAs. The RNA Pol I promoter is known to be species-specific. The RNA Pol I promoter from one species may not be recognized by RNA Pol I enzymes from distantly related species. Until now, human, dog, chicken and mouse derived RNA Pol I promoter have been cloned and used for constructing influenza virus reverse genetics system. However, up to now, the studies on the horse RNA Pol I promoter have not been reported. Accordingly, many researches on the influenza virus can not be performed in horse-derived cells. Considering this, based on the location characteristic of the eukaryote species, the horse RNA Pol I transcription initiation site is identified in the equus caballus genome database in the Gen Bank and ensembl database. The horse RNA Pol I promoter sequence is acquired by PCR, using the genomic DNA extracted from fetal equine lung cells. Using the minigenome replication assay based on the horse RNA Pol I promoter of different lengths, it is observed that a 500 bp horse RNA Pol I promoter sequence is required for transcription efficiency. The difference between the polymerase activities of two equine influenza viruses is compared by the constructed minigenome replication assay, and the PA protein is identified playing a key role. The influence of Mx A protein on the polymerase activity of influenza virus is also identied. Finally, using the developed reverse genetics system based on the horse RNA Pol I promoter, wild and recombinant equine influenza virus is generated. The successful clone and application of the horse RNA Pol I promoter enriches our knowledge of the RNA Pol I promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells. The constructed reverse genetics system will contribute to the influenza virus study in horse cells.The recent studies suggest all of the human, pig, chicken, duck and mouse IFITM proteins have antiviral activity. Although equine is an important host of influenza virus,no study has been performed on the influence of horse IFITM protein on blocking influenza virus. In our study, the IFITM gene is searched in the Gen Bank database and specific primers for it is designed. Six horse IFITM genes are cloned by RT-PCR. Firstly,the expression characteristic of horse IFITM m RNA is studied. The expression level of the IFITM m RNA in the horse cell and tissue is detected by real-time PCR. After treated with interferon or infected with influenza virus, the expression level of the IFITM m RNA in the horse cell is up-regulated. However, after infected with herpesvirus, the expression level of the IFITM m RNA in the horse cell is not changed. Next, the celluar localization of horse IFITM protein is investigated. The eukaryotic expression plasmids expressing horse IFITM are constructed. The IFITM protein is detected successfully by indirect immunofluorescence assay and Western blot. Detected by laser scanning confocal microscopy, the celluar localization of horse IFITM is different between each other. The horse IFITM3 protein is co-localizated with LAMP1 protein and the other horse IFITM proteins are not co-localizated with LAMP1 protein. Finally, using transient transfection with eukaryotic expression plasmids and cell lines stablely expressing proteins, the bocking influenza virus activity of horse IFTM is determined. Through the lentivirus infection, the MDCK lines expressing the horse IFITM protein are successfully established. And the expression of horse IFITM protein is successfully detected by indirect immunofluorescence assay and Western blot. The blocking influenza virus activity of the horse IFITM is confirmed by both transient transfection and stable transfection by the relative luciferase assay based on horse or dog RNA Pol I promoter. The study on the horse IFITM protein enriches our knowledge of the blocking function of IFITM protein and contributes to the development of antiviral drug.Since 2012, three novel viruses, known as equine hepatitis C virus(EHCV), equine pegivirus(EPg V) and Theiler’s Disease Associated Virus(TDAV), have been discovered in equines as the newest members of the Flaviviridae family. The genomic information concerning circulating equine NPHV, EPg V and TDAV strains around the world is limited. To date, no genetic surveillance studies have been performed on these three novel viruses in the equine population of China. Here, a total of 177 serum samples were collected from equines across China between 2014 and 2015. Using PCR, we detected viral RNA in the serum samples, six of which were NPHV-positive, and two of which were EPg V-positive. Co-infection of the two viruses was not observed among equines in China. TDAV RNA was not detected in the equine serum samples collected for this study. Phylogenetic analysis of partial NS5 B, NS3, and 5’UTR nucleotide sequences from equine NPHV, as well as partial NS3 nucleotide sequences from EPg V, indicated that equine NPHV and EPg V have evolved into two main clades, both of which are circulating in China. This study enriches our knowledge of the geographic distribution and genetic evolution of these three novel equine viruses. |
