H5N1 influenza represents one of the great challenges to public health.  Some H5N1 viruses (i.e., A/goose/Hubei/65/05, GS/65) are weakly pathogenic, while the others (i.e., A/duck/Hubei/49/05, DK/49) are highly pathogenic to their natural hosts.  Here, we performed brain and spleen transcriptomic analyses of control ducks and ones infected by the DK/49 or the GS/65 H5N1 virus.  We demonstrated that, compared to the GS/65 virus, the DK/49 virus infection changed more numerous immune genes’ expression and caused continuous increasing of immune pathways (i.e., RIG-I and MDA5) in ducks.  We found that both H5N1 virus strains might escape or subvert host immune response through affecting alternative translation of immune genes, while the DK/49 virus seemed to induce alternative translation of more immune genes than the GS/65 virus.  We also identified five co-expressional modules associated with H5N1 virus replication through the weight correlation network analysis (WGCNA).  Moreover, we first demonstrated that the duck BCL2L15 and DCSTAMP in one of these five modules inhibited both the highly pathogenic and weakly pathogenic H5N1 virus replication efficiently.  These analyses, in combination with our comprehensive transcriptomic data, provided global view of the molecular architecture for the interaction between host and H5N1 viruses. 

2´-5´-Oligoadenylate synthetase like protein (OASL) plays a key role in response to viral infections through selectively activating the OAS/RNase L or OASL/RIG-I signaling pathway.  Although classic pathway of OASL is well-known, its regulated genes or co-actors are largely unknown.  To study the possible molecular mechanism of duck OASL (dOASL), we performed RNA-sequencing (RNA-seq) and immunoprecipitation and mass spectrometry (IP-MS) at the level of mRNA and protein, respectively.  For RNA-seq, we used DF1 cell lines (DF1^dOASL+/+, DF1^{cOASL–/–,} and DF1) with or without the CK/0513 H5N1 virus (A/chicken/huabei/0513/2007) infection.  1 737 differentially expressed genes (DEGs) were identified as candidate target genes regulated by dOASL.  Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Weighted Correlation Network Analysis (WGCNA) were performed.  We identified one important yellow co-expression module correlated with antiviral immune response.  In this module, Ankyrin repeat and FYVE domain containing 1 (ANKFY1), harboring a BTB domain similar to the methyl CpG-binding protein 1 (MBD1) which bound to OASL in human, was regulated by dOASL.  At protein level, 133 host proteins were detected.  Interestingly, ANKFY1 was one of them binding to dOASL protein.  Further phylogenomic and chromosomal syntenic analysis demonstrated MBD1 was absent in birds, while mammals retained.  It is suggested that OASL-ANKFY1 interaction might act as a compensatory mechanism to regulate gene expression in birds.  Our findings will provide a useful resource for the molecular mechanism research of dOASL.

CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms, but its use in pigs to generate precise genetic modifications has seldom been described.  Myostatin (MSTN) is a well-characterized negative regulator of muscle development, and natural mutations in this gene cause a double-muscled phenotype in many species.  However, to the best of our knowledge, no naturally occurring mutation in MSTN has been found in pigs.  In addition, no living pig models with sophisticated modifications orthologous to natural mutations in MSTN have yet been reported.  In this study, we exploited the CRISPR/Cpf1 system to introduce a predefined modification orthologous to the natural MSTN mutation found in Belgian Blue cattle (thus known as the Belgian Blue mutation).  Our research demonstrated that the cutting efficiency of CRISPR/Cpf1 was 12.3% in mixed porcine fetal fibroblasts in drug free medium, and 41.7% in clonal colonies obtained using G418 selection.  Then, the Cpf1-sgRNA vector, ssODN template, and a self-excision cassette were co-transfected into porcine fetal fibroblasts.  After G418 selection, 8 clonal colonies were examined and 5 with genetic modification were found.  Of these 5, 2 harbored the precise 11-bp deletion.  Using 1 heterozygous clonal colony, 2 cloned Duroc piglets were successfully generated, which was heterozygous for the Belgian Blue mutation.  In summary, our results demonstrate that CRISPR/Cpf1 system can be used efficiently to generate double-stranded breaks, and also to mediate homologous recombination to introduce precise genomic modifications in pigs.