Development Of Protective Monoclonal Antibodies Against H3N2 Canine Influenza Virus And The Effect Of Cellular Microrna On Virus Replication

H3N2 canine influenza virus (CIV) is a highly contagious disease that is popular among dog populations, which can cause dogs cough, fever, pneumonia and even death. CIV not only leads to severe harm to the survival and health of dogs, but brings a huge risk to human health as well. If possible to develop effective monoclonal antibody(mAb) against canine influenza virus, and to clarify the infection mechanism of CIV, will undoubtedly efficiently large contribute to the prevention and control of CIV infection. However till now, there is no mAb direct to H3N2 CIV reported, nor has suitable cell model for CIV study as well. Thus, our present study developed protective H3N2 CIV mAb and established immortalized canine bronchial epithelial cells to be used as cell model for CIV study. In addition, we obtained differentiated expression profiles after primary canine bronchial epithelial cells and primary alveolar macrophages being infected with H3N2 CIV. Among them, three microRNAs were selected to investigate their function after viral infection. Study results demonstrated that after host cells being infected with CIV, host has the capacity to influence virus replication in related cells by changing the expression levels of microRNA themselves. The findings shed light on clarifying host cell molecular mechanisms after canine influenza virus infection.1. The development of monoclonal antibodies against H3N2 canine influenza virus and their characteristics analysisIn this study, H3N2 canine influenza virus strain A/Canine/Jiangsu/06/2010 (JS/10)was being amplified, and purified whole virus was used as antigen for immunizing BALB/c mice with multi-point intraderma injection. After cell fusion and subclone of hybridoma,we totally obtained 7 hybridoma cells which were capable of secreting monoclonal antibody, then ascites being developed and purified of these 7 hybridoma cells, eventually 7 monoclonal antibodies were successfully got and their characteristics being analyzed. Of the 7 monoclonal antibodies, one of which named monoclonal antibody D7 whose neutralizing titer was 1: 12800 and cross-reactivity to different influenza virus strains of H3N2 subtype were all positive. In addition, western blot identification results showed that the epitope of this monoclonal antibody was located in relatively conserved HA2 fragment.In summary,the development of H3N2 canine influenza virus monoclonal antibodies in the present study not only provides important material for the diagnosis and detection of the virus, but provides a drug candidate for the treatment of viral infection as well, which has potential application value.2. Monoclonal antibody protection against H3N2 influenza virus strains after virus infectionTo assess the protective effect of the mAb against H3N2 CIV infection, and at the same time to make it clear that whether the mAb could have cross-protection effect on different influenza virus strains of subtype H3N2 or not, in this study, BALB/c mice infection model was used to evaluate protective effect of mAb D7 against H3N2 CIV isolate JS /10 solated in Jiangsu Province, H3N2 CIV isolate GD/12 isolated from Guangdong Province and H3N2 swine influenza virus isolate SD/05 isolated from Shandong Province. One day after immunization mAb D7 and irrelevant mAb in mice,three influenza virus isolates mentioned above were used to infect mice, irrelevant mAb group, separate viral infection group and PBS group were used as control, five time points which was 2,4,6,10 and 14 day post infection, we measured changes in body weight,collected blood, feces and different organs to test viral load changes, and lung tissues of mice used for cytokines (IFN-? and TNF-? ) expression level comparison among different infection groups and control groups. We also chose heart, lung, brain these three organs of mice in different infection and control groups for immunohistochemistry test. The data showed that mAb D7 protected mice from infection with the three virus strains, especially the homologous strain, which was indicated by the recovery of body weight, reduction of viral load, and reduction of tissue damage. Moreover, the levels of IFN-? and TNF-? in the lungs, as detected by ELISA, were reduced in the infected mice treated with the mAb D7 compared with those without mAb D7 treatment. Thus, our findings demonstrate, for the first time,that a mAb could reduce the release of IFN-? and TNF-? associated with tissue damage by CIV infection and that the mAb might be of great therapeutic value for CIV infection.3. Establishment of immortalized canine bronchiolar epithelial cells and its characteristics analysisHere, we cultured primary canine bronchiolar epithelial cells (CBECs), whose characteristics were confirmed by their expression of the epithelial cell-specific marker cytokeratin 18, and have provided protocols for their isolation and ex vivo expansion.Further, we established immortalized CBECs containing the human telomerase reverse transcriptase (hTERT) gene via transfection of primary CBECs with the recombinant plasmid pEGFP-hTERT. Immortalized bronchiolar epithelial cells (hTERT-CBECs) retain the morphological and functional features of primary CBECs,as indicated by reverse transcriptase polymerase chain reaction, proliferation assays, karyotype analysis,telomerase activity assay, and western blot, whichdemonstrate that hTERT-CBECs have higher telomerase activity, an extended proliferative lifespan, and a diploid complement of chromosomes,even after Passage 50. Moreover, this cell line is not transformed,as evaluated using soft agar assays and tumorigenicity analysis in nude mice, and can therefore be safely used in future studies. The isolation and establishment of stable hTERT-CBECs is of great importance for use as an in vitro model for mechanistic studies of canine pathogenic infections.4. MicroRNA expression profile analysis of primary canine bronchiolar epithelial cells and primary canine alveolar macrophages after CIV infectionThis study firstly isolated and cultured primary canine bronchial epithelial cells and primary canine alveolar macrophages, and by small RNA sequencing and microarray microRNA sequencing,we received differential expression profiles of the two types of cells before and after infection with H3N2 canine influenza virus. Results showed that 421 and 292 microRNA (miRNA) expression levels were detected in primary canine bronchial epithelial cells and primary canine alveolar macrophages, respectively. Of them, 117 and 71 differentially expressed miRNAs that fold change more than 2 were selected, respectively.By using TargetScan, PITA and miRanda database for target gene prediction of these differentially expressed miRNAs, and Gene Ontology and KEGG database were used for function prediction and annotation on these target genes of differentially expressed miRNAs. From results,we found these predicted miRNAs together with their potential target genes were mainly involved in anti-viral response, apoptosis response, inflammatory factor response like chemokines and cytokines and other signaling pathways. In addition,sequencing results were verified by representative miRNAs qRT-PCR and found qRT-PCR results showed consistent expression levels compared with results of miRNA sequencing.In conclusion, differential miRNA expression profiles obtained in this study have provided information foundation in studying pathogenesis of canine influenza virus, and will help to clarify the mechanisms of cellular defense mechanism against canine influenza virus infection. While these selected miRNAs may become the drug candidate targets for treatment of canine influenza virus, which will contribute to prevention and control of this virus.5. Effect of host cell microRNA to H3N2 canine influenza virus replicationThis study chose three miRNAs which were significantly down regulation, which were cfa-miR-125b, cfa-miR-151 and cfa-miR-423a, respectively, and investigated their cellular response roles against viral infection. On basis of previous established immortalized canine bronchial epithelial cells (hTERT-CBECs) and canine macrophage cell line (DH82), we constructed three miRNAs overexpression and inhibition stable cell lines by using adenoviral vectors and lentiviral vectors, respectively. Then we assessed virus replication capacity through TCID50, at the same time tested NP and NS1 expression levels on the three miRNAs overexpression and inhibition related infected cell lines through real-time quantitative PCR (qRT-PCR) and western blot test. Results showed that when overexpression cfa-miR-125b and cfa-miR-151, virus proliferation capacity increased,while these two miRNAs expression were inhibited, virus replication significantly declined.However, overexpression or inhibition of cfa-miR-423a has no significant influence to virus replication. In additon, qRT-PCR and western blot results also showed that both the overexpression and inhibition of cfa-miR-125b and cfa-miR-151 were able to raise and lower the viral NP expression level. The overexpression and inhibition of cfa-miR-125b of could also raise and lower the expression level of NS1, however, the overexpression of cfa-miR-151 had no effect on the viral NS1 expression level. This phenomenon existed in both hTERT-CBECs and DH82 cells. In addition, both of the overexpression and inhibition of cfa-miR-423a could decrease the expresson level of NP, but had no effect on the expression level of NS1. Our study demonstrated that when cells were infected with H3N2 canine influenza virus, virus replication can be regulated by changing miRNA level themselves, which can help us further elucidate the mechanism of cellar defense to viral infection.