Sequential disruption of ALV host receptor genes reveals no sharing of receptors between ALV subgroups A, B, and J

Background: Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus(ALV)subgroups using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A(TVA) and generate chicken cells resistant to infection by this virus.Results: CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing. Conversely,overexpression of the wild-type TVA receptor(wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na^+/H^+ exchange 1(chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively.Conclusions: Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell entry.

Herpesvirus of Turkeys (Meleagridis Herpesvirus 1) Encodes a Functional MicroRNA-221 Homolog with High Sequence Conservation

Herpesviruses account for most of the known virus-encoded miRNAs. Herpesvirus of turkey (HVT), a non-pathogenic avian herpesvirus used as an avian vaccine and viral vector, encodes 28 mature miRNAs. This included HVT-miR-H14-3p that showed almost identical sequence to gga-miR-221, suggesting that it is pirated from the avian host. Although the functional homolog between the two miRNAs has been proposed based on the sequence similarity, the direct experimental evidence is still lacking. In this report, we provide the evidence for the first time that HVT-miR-H14-3p is indeed a gga-miR-221 homolog through modulating the expression of p27Kip1, a known target of miR-221 by binding to its 3’UTR. We also created an HVT-miR-H14-3p deletion virus and show that this miRNA is not essential for in vitro replication.

Critical roles of non-coding RNAs in lifecycle and biology of Marek's disease herpesvirus

Over the past two decades, numerous non-coding RNAs(ncRNAs) have been identified in different biological systems including virology, especially in large DNA viruses such as herpesviruses. As a representative oncogenic alphaherpesvirus, Marek’s disease virus(MDV) causes an important immunosuppressive and rapid-onset neoplastic disease of poultry, namely Marek’s disease(MD). Vaccinations can efficiently prevent the onset of MD lymphomas and other clinical disease, often heralded as the first successful example of vaccination-based control of cancer. MDV infection is also an excellent model for research into virally-induced tumorigenesis. Recently, great progress has been made in understanding the functions of ncRNAs in MD biology.Herein, we give a review of the discovery and identification of MDV-encoded viral miRNAs, focusing on the genomics,expression profiles, and emerging critical roles of MDV-1 miRNAs as oncogenic miRNAs(oncomiRs) or tumor suppressor genes involved in the induction of MD lymphomas. We also described the involvements of host cellular miRNAs, lincRNAs, and circRNAs participating in MDV life cycle, pathogenesis, and/or tumorigenesis. The prospects, strategies, and new techniques such as the CRISPR/Cas9-based gene editing applicable for further investigation into the ncRNA-mediated regulatory mechanisms in MDV pathogenesis/oncogenesis were also discussed, together with the possibilities of future studies on antiviral therapy and the development of new efficient MD vaccines.