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In the past decade, research has demonstrated that viral miRNAs encoded by a number of viral genomes, particularly by most of the herpesvirus including Marek's disease virus (MDV), play important regulatory roles in viral infection, replication, and regulation of tumorigenesis. As macrovesicles in cells, exosomes can deliver viral miRNAs and exert gene regulatory functions. Whether the exosomes play a role in the replication, pathogenesis/tumorigenesis of avian herpesviruses such as oncogenic Marek's disease virus (MDV) remains unclear. Herein we extracted and identified the exosomes from MDV-transformed T cell line MSB-1 and demonstrated high abundance of MDV-1 miRNA expression. Using dual luciferase-based reporter assay, we also demonstrated that the exosomes derived from MSB-1 can deliver functional miRNA successfully into primary chicken embryo fibroblasts. These findings provide new insights into the role of exosomes and the mechanisms of how virus-encoded miRNA function in MDV latency/activation switching, viral replication, pathogenesis and/or tumorigenesis.

ABSTRACT: Avian leukosis virus subgroup J (ALV-J) is a retrovirus that can cause immunosuppression and tumors in chicken. However, relative pathogenesis is still not clear. At present, metabolomics has shown great potential in the screening of tumor metabolic markers, prognostic evaluation, and drug target design. In this study, we utilize an untargeted metabolomics approach based on ultrahigh-performance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) to analyze the metabolic changes in chicken embryo fibroblast (CEF) cells infected by ALV-J. We found that ALV-J infection significantly altered a wealth of metabolites compared with control group. Additionally, most of the differentially expressed metabolites belonged to lipid metabolism, purine nucleotide metabolism and amino acid metabolism. Among them, the proportion of lipid metabolites account for the highest proportion (around 31%). Results suggest that these changes may be conductive to the formation of virion, thereby promoting the replication of ALV-J. These data provided metabolic evidence and potential biomarkers for the cellular metabolic changes induced by ALV-J, and provided important insight for further understanding the replication needs and pathogenesis of ALV-J.

Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs’ eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek’s disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.

Poultry enteric bacterial diseases are of significant economic importance because they are responsible for production losses due to weight loss, increased morbidity and mortality, and increased cost of production arising from poor feed conversion and treatment. This cross-sectional purposive study characterized enteric bacterial pathogens in poultry from selected agroclimatic regions in Kenya and investigated their antimicrobial resistance gene profiles. Cloacal (n = 563) and oropharyngeal (n = 394) swabs were collected and pooled into 16 and 14 samples, respectively, to characterize bacterial pathogens and their antimicrobial resistance gene profiles. We report that Proteobacteria, Chlamydiae, and Firmicutes are the most dominant phyla present in both cloacal and oropharyngeal swabs of the six poultry species studied, indicating the colonization of the poultry gut by many pathogenic bacteria. Using KEGG and COG databases, some pathways related to metabolism, genetic information, and cellular processing were detected. We also report the abundance of antimicrobial resistance genes that confer resistance to β-lactamases, aminoglycosides, and tetracycline in most of the poultry analyzed, raising concern about the dangers associated with continuous and inappropriate use of these antibiotics in poultry production. The antimicrobial resistance gene data generated in this study provides a valuable indicator of the use of antimicrobials in poultry in Kenya. The information generated is essential for managing bacterial diseases, especially in backyard poultry raised under scavenging conditions.

Chicken lung is an important target organ of avian influenza virus (AIV) infection, and different pathogenic virus strains lead to opposite prognosis. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of 16 cell types (19 clusters) in the lung tissue of chickens infected with H5N1 highly pathogenic avian influenza virus (HPAIV) and H9N2 low pathogenic avian influenza virus (LPAIV), respectively. Notably, we developed a valuable catalog of marker genes for these cell types. Compared to H9N2 AIV infection, H5N1 AIV infection induced extensive virus replication and the immune reaction across most cell types simultaneously. More importantly, we propose that infiltrating inflammatory macrophages (clusters 0, 1, and 14) with massive viral replication, pro-inflammatory cytokines (IFN-β, IL1β, IL6 and IL8), and emerging interaction of various cell populations through CCL4, CCL19 and CXCL13, potentially contributed to the H5N1 AIV driven inflammatory lung injury. Our data revealed complex but distinct immune response landscapes in the lung tissue of chickens after H5N1 and H9N2 AIV infection, and deciphered the potential mechanisms underlying AIV-driven inflammatory reactions in chicken. Furthermore, this article provides a rich database for the molecular basis of different cell-type responses to AIV infection.

Fowl cholera is caused by the bacterium Pasteurella multocida, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines that are still in use today despite their many limitations. In this research, we describe the development of a genetically engineered herpesvirus of turkeys (HVT) that carries the OmpH gene from P. multocida integrated into UL 45/46 intergenic region using CRISPR/Cas9-NHEJ and Cre-Lox system editing. The integration and expression of the foreign cassettes were confirmed using polymerase chain reaction (PCR), indirect immunofluorescence assays, and Western blot assays. The novel recombinant virus (rHVT-OmpH) demonstrated stable integration of the OmpH gene even after 15 consecutive in vitro passages, along with similar in vitro growth kinetics as the parent HVT virus. The protective efficacy of the rHVT-OmpH vaccine was evaluated in vaccinated ducks by examining the levels of P. multocida OmpH-specific antibodies in serum samples using ELISA. Groups of ducks that received the rHVT-OmpH vaccine or the rOmpH protein with Montanide™ (SEPPIC, Paris, France) adjuvant exhibited high levels of antibodies, in contrast to the negative control groups that received the parental HVT or PBS. The recombinant rHVT-OmpH vaccine also provided complete protection against exposure to virulent P. multocida X-73 seven days post-vaccination. This outcome not only demonstrates that the HVT vector possesses many characteristics of an ideal recombinant viral vaccine vector for protecting non-chicken hosts, such as ducks, but also represents significant research progress in identifying a modern, effective vaccine candidate for combatting ancient infectious diseases.

ABSTRACT Marek’s disease virus (MDV) induces immunosuppression and neoplastic disease in chickens. The virus is controllable via an attenuated meq deletion mutant virus, which has the disadvantage of retaining the ability to induce lymphoid organ atrophy. To overcome this deficiency and produce more vaccine candidates, a recombinant MDV was generated from the highly virulent Md5BAC strain, in which both meq and a cytolytic replication-related gene, pp38, were deleted. Replication of the double deletion virus, Md5BAC ΔmeqΔpp38, was comparable with that of the parental virus in vitro. The double deletion virus was shown to be fully attenuated and to reduce lymphoid organ atrophy in vivo. Crucially, Md5BAC ΔmeqΔpp38 confers superior protection against highly virulent virus compared with a commercial vaccine strain, CVI988/Rispens. Transcriptomic profiling indicated that Md5BAC ΔmeqΔpp38 induced a different host immune response from CVI988/Rispens. In summary, a novel, effective, and safe vaccine candidate for prevention and control of MD caused by highly virulent MDV is reported. IMPORTANCE MDV is a highly contagious immunosuppressive and neoplastic pathogen. The virus can be controlled through vaccination via an attenuated meq deletion mutant virus that retains the ability to induce lymphoid organ atrophy. In this study, we overcame the deficiency by generating meq and pp38 double deletion mutant virus. Indeed, the successfully generated meq and pp38 double deletion mutant virus had significantly reduced replication capacity in vivo but not in vitro. It was fully attenuated and conferred superior protection efficacy against very virulent MDV challenge. In addition, the possible immunological protective mechanism of the double deletion mutant virus was shown to be different from that of the gold standard MDV vaccine, CVI988/Rispens. Overall, we successfully generated an attenuated meq deletion mutant virus and widened the range of potential vaccine candidates. Importantly, this study provides for the first time the theoretical basis of vaccination induced by fully attenuated gene-deletion mutant virus.

Avian leukosis caused by avian leukosis virus (ALV), belonging to the genus Alpharetrovirus of the family Retroviridae, is associated with benign and malignant tumors in hemopoietic cells in poultry. Although several methods have been developed for ALV detection, most of them are not suitable for rapid on-site testing due to instrument limitations, professional operators, or the low sensitivity of the method. Herein, we described the real-time recombinase polymerase amplification (RPA) assay for rapid detection of ALV subgroup J (ALV-J). The major viral structural glycoprotein gp85, highly specific for the subgroup, was used as the molecular target for the real-time RPA assay. The results were obtained at 38°C within 20 min, with the detection sensitivity of 10 copies/μl of standard plasmid pMD18-T-gp85 as the template per reaction. Real-time RPA was capable of ALV-J-specific detection without cross-reaction with other non-targeted avian pathogens. Of the 62 clinical samples tested, the ALV-positive rates of real-time RPA, PCR, and real-time PCR were 66.13% (41/62), 59.68% (37/62), and 67.74% (42/62), respectively. The diagnostic agreement between real-time RPA and real-time PCR was 98.39% (61/62), and the kappa value was 0.9636. The developed real-time ALV-J assay seems promising for rapid and sensitive detection of ALV-J in diagnostic laboratories. It is suitable for on-site detection, especially in a poor resource environment, thus facilitating the prevention and control of ALV-J.

Marek’s disease (MD) caused by pathogenic Marek’s disease virus type 1 (MDV−1) is one of the most important neoplastic diseases of poultry. MDV−1-encoded unique Meq protein is the major oncoprotein and the availability of Meq-specific monoclonal antibodies (mAbs) is crucial for revealing MDV pathogenesis/oncogenesis. Using synthesized polypeptides from conserved hydrophilic regions of the Meq protein as immunogens, together with hybridoma technology and primary screening by cross immunofluorescence assay (IFA) on Meq-deleted MDV−1 viruses generated by CRISPR/Cas9-gene editing, a total of five positive hybridomas were generated. Four of these hybridomas, namely 2A9, 5A7, 7F9 and 8G11, were further confirmed to secrete specific antibodies against Meq as confirmed by the IFA staining of 293T cells overexpressing Meq. Confocal microscopic analysis of cells stained with these antibodies confirmed the nuclear localization of Meq in MDV-infected CEF cells and MDV-transformed MSB-1 cells. Furthermore, two mAb hybridoma clones, 2A9-B12 and 8G11-B2 derived from 2A9 and 8G11, respectively, displayed high specificity for Meq proteins of MDV−1 strains with diverse virulence. Our data presented here, using synthesized polypeptide immunization combined with cross IFA staining on CRISPR/Cas9 gene-edited viruses, has provided a new efficient approach for future generation of specific mAbs against viral proteins.

Abstract Processing and packaging of herpesvirus genomic DNA is regulated by a packaging-associated terminase complex comprising of viral proteins pUL15, pUL28 and pUL33. Marek’s disease virus (MDV) homologs UL28 and UL33 showed conserved functional features with high sequence identity with the corresponding Herpes simplex virus 1 (HSV-1) homologs. As part of the investigations into the role of the UL28 and UL33 homologs of oncogenic MDV for DNA packaging and replication in cultured cells, we generated MDV mutant clones deficient in UL28 or UL33 of full-length MDV genomes. Transfection of UL28- or UL33-deleted BAC DNA into chicken embryo fibroblast (CEF) did not result either in the production of visible virus plaques, or detectable single cell infection after passaging onto fresh CEF cells. However, typical MDV plaques were detectable in CEF transfected with the DNA of revertant mutants where the deleted genes were precisely reinserted. Moreover, the replication defect of the UL28-deficient mutant was completely restored when fragment encoding the full UL28 gene was co-transfected into CEF cells. Viruses recovered from the revertant construct, as well as by the UL28 co-transfection, showed replication ability comparable with parental virus. Furthermore, the transmission electron microscopy study indicated that immature capsids were assembled without the UL28 expression, but with the loss of infectivity. Importantly, predicted three-dimensional structures of UL28 between MDV and HSV-1 suggests conserved function in virus replication. For the first time, these results revealed that both UL28 and UL33 are essential for MDV replication through regulating DNA cleavage and packaging.

Turkey herpesvirus (HVT) has been widely used as a successful live virus vaccine against Marek's disease (MD) in chickens for more than five decades. Increasingly, HVT is also used as a highly effective recombinant vaccine vector against multiple avian pathogens. Conventional recombination, or recombineering, techniques that involve the cloning of viral genomes and, more recently, gene editing methods have been used for the generation of recombinant HVT-based vaccines. In this study, we used NHEJ-dependent CRISPR/Cas9-based approaches to insert the mCherry cassette for the screening of the HVT genome and identifying new potential sites for the insertion of foreign genes. A novel intergenic site HVT-005/006 in the unique long (UL) region of the HVT genome was identified, and mCherry was found to be stably expressed when inserted at this site. To confirm whether this site was suitable for the insertion of other exogenous genes, haemagglutinin (HA) of the H9N2 virus was inserted into this site, and a recombinant HVT-005/006-HA was rescued. The recombinant HVT-HA can grow well and express HA protein stably, which demonstrated that HVT-005/006 is a promising site for the insertion of foreign genes.

Reticuloendotheliosis virus (REV) is an avian retrovirus that causes an oncogenic, immunosuppressive, and runting-stunting syndrome in avian hosts. The co-infection of REV and Marek’s disease virus (MDV), an oncogenic herpesvirus in chickens, further increases disease severity and reduces MDV vaccine efficacy. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has successfully been used against pathogens in mammalian cells. However, the large size of the CRISPR-Cas9 coding sequences makes its in vivo delivery challenging. Here, following the design of a panel of single-guided RNAs targeting REV, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the long terminal repeats of REV, resulting in the inhibition of viral protein expression. The CRISPR-Cas9 system disrupts the integrated proviral genome and provides defense against new viral infection and replication in chicken cells. Moreover, by constructing recombinant MDV carrying CRISPR-Cas9 components using an attenuated MDV vaccine strain as the vector, we efficiently delivered the CRISPR-Cas9 system into chickens, and the MDV-delivered CRISPR-Cas9 drastically reduced REV viral load and significantly diminished REV-associated symptoms. To our knowledge, this is the first study establishing avian retrovirus resistance in chickens utilizing herpesvirus-delivered CRISPR-Cas9, which provides a novel and effective strategy against viral infections.

The avian immunosuppressive and neoplastic diseases caused by Marek’s disease virus (MDV), avian leucosis virus (ALV), and reticuloendotheliosis virus (REV) are seriously harmful to the global poultry industry. In recent years, particularly in 2020–2022, outbreaks of such diseases in chicken flocks frequently occurred in China. Herein, we collected live diseased birds from 30 poultry farms, out of 42 farms with tumour-bearing chicken flocks distributed in central China, to investigate the current epidemiology and co-infections of these viruses. The results showed that in individual diseased birds, the positive infection rates of MDV, ALV, and REV were 69.5% (203/292), 14.4% (42/292), and 4.7% (13/277), respectively, while for the flocks, the positive infection rates were 96.7% (29/30), 36.7% (11/30), and 20% (6/30), respectively. For chicken flocks, monoinfection of MDV, ALV, or REV was 53.3% (16/30), 3.3% (1/30), and 0% (0/30), respectively, but a total of 43.3% (13/30) co-infections was observed, which includes 23.3% (7/30) of MDV+ALV, 10.0% (3/30) of MDV+REV, and 10.0% (3/30) of MDV+ALV+REV co-infections. Interestingly, no ALV+REV co-infection or REV monoinfection was observed in the selected poultry farms. Our data indicate that the prevalence of virulent MDV strains, partially accompanied with ALV and/or REV co-infections, is the main reason for current outbreaks of avian neoplastic diseases in central China, providing an important reference for the future control of disease.

Turkey herpesvirus (HVT) is widely used as an effective recombinant vaccine vector for expressing protective antigens of multiple avian pathogens from different loci of the HVT genome. These include the HVT029/031 (UL22–23) locus for the insertion of IBDV VP2 and the recently identified HVT005/006 locus as a novel site for expressing heterologous proteins. In order to compare the efficacy of recombinant vaccines with the HA gene at different sites, the growth curves and the HA expression levels of HVT-005/006-hCMV-HA, HVT-005/006-MLV-HA, and HVT-029/031-MLV-HA were first examined in vitro. While the growth kinetics of three recombinant viruses were not significantly different from those of parent HVT, higher expression of the HA gene was achieved from the HVT005/006 site than that from the HVT029/031 site. The efficacy of the three recombinant viruses against avian influenza H9N2 virus was also evaluated using one-day-old SPF chickens. Chickens immunized with HVT-005/006-MLV-HA or HVT-005/006-hCMV-HA displayed reduced virus shedding compared to HVT-029/031-MLV-HA vaccinated chickens. Moreover, the overall hemagglutination inhibition (HI) antibody titers of HVT-005/006-HA-vaccinated chickens were higher than that of HVT-029/031-HA-vaccinated chickens. However, HVT-005/006-MLV-HA and HVT-005/006-hCMV-HA did not result in a significant difference in the level of HA expression in vitro and provided the same protective efficacy (100%) at 5 days after challenge. In the current study, the results suggested that recombinant HVT005/006 vaccines caused better expression of HA than recombinant HVT029/031 vaccine, and that HVT-005/006-MLV-HA or HVT-005/006-hCMV-HA could be a candidate vaccine for the protection of chickens against H9N2 influenza.

Marek’s disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek’s disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant—GX0101Δpp38—and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas—namely, 4F9, 31G7, 34F2, and 35G9—were demonstrated to secrete specific antibodies against MDV-1’s pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography–tandem mass spectrometry (LC–MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses—especially for large DNA viruses such as herpesviruses.

Abstract Background Avian leukosis (AL), which is caused by avian leukosis virus (ALV), has led to substantial economic losses in the poultry industry. The kit used to detect all ALV-positive chickens in breeder flocks is very important for efficiently controlling AL. However, a new emerging ALV subtype is currently a severe challenge in the poultry industry. Results In this paper, we compared different enzyme-linked immunosorbent assay (ELISA) kits for detecting p27 of ALV in the same batch of meconium samples. Different positive samples were further analyzed by PCR or virus isolation. The results showed that 36 positive samples among the 1812 chicken meconium samples could be detected by a sandwich ELISA (sELISA) kit, but only 17 positive samples could be identified by a commercial kit. To verify this result, cloacal swabs and viruses isolated from the positive chickens (2 days old) were used to detect the presence of p27. The results showed that the positive rate of p27 was 100% for the swabs and 40% for virus isolation. Surprisingly, PCR and sequence analysis revealed that the env gene of ALV in these positive samples belonged to the novel subgroup K (ALV-K). Conclusion These data not only demonstrate the relatively high sensitivity of the sELISA kit but also highlight the challenge of controlling ALV-K.

Abstract 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.

Duck enteritis virus and Pasteurella multocida are major duck pathogens that induce duck plague and fowl cholera, respectively, in ducks and other waterfowl populations, leading to high levels of morbidity and mortality. Immunization with live attenuated DEV vaccine containing P. multocida outer membrane protein H (OmpH) can provide the most effective protection against these two infectious diseases in ducks. We have recently reported the construction of recombinant DEV expressing P. multocida ompH gene using the CRISPR/Cas9 gene editing strategy with the goal of using it as a bivalent vaccine that can simultaneously protect against both infections. Here we describe the findings of our investigation into the systemic immune responses, potency and clinical protection induced by the two recombinant DEV-ompH vaccine constructs, where one copy each of the ompH gene was inserted into the DEV genome at the UL55-LORF11 and UL44-44.5 intergenic regions, respectively. Our study demonstrated that the insertion of the ompH gene exerted no adverse effect on the DEV parental virus. Moreover, ducklings immunized with the rDEV-ompH-UL55 and rDEV-ompH-UL44 vaccines induced promising levels of P. multocida OmpH-specific as well as DEV-specific antibodies and were completely protected from both diseases. Analysis of the humoral and cellular immunity confirmed the immunogenicity of both recombinant vaccines, which provided strong immune responses against DEV and P. multocida. This study not only provides insights into understanding the immune responses of ducks to recombinant DEV-ompH vaccines but also demonstrates the potential for simultaneous prevention of viral and bacterial infections using viral vectors expressing bacterial immunogens.

In recent years, outbreaks of Marek’s disease (MD) have been frequently reported in vaccinated chicken flocks in China. Herein, we have demonstrated that four Marek’s disease virus (MDV) isolates, HN502, HN302, HN304, and HN101, are all pathogenic and oncogenic to hosts. Outstandingly, the HN302 strain induced 100% MD incidence, 54.84% mortality, and 87.10% tumor incidence, together with extensive atrophy of immune organs. Pathotyping of HN302 was performed in comparison to a standard very virulent (vv) MDV strain Md5. We found that both CVI988 and HVT vaccines significantly reduced morbidity and mortality induced by HN302 or Md5 strains, but the protection indices (PIs) provided by these two vaccines against HN302 were significantly lower (27.03%) or lower (33.33%) than that against Md5, which showed PIs of 59.89% and 54.29%, respectively. These data suggested that HN302 possesses a significant higher virulence than Md5 and at least could be designated as a vvMDV strain. Together with our previous phylogenetic analysis on MDV-1 meq genes, we have presently suggested HN302 to be a typical highly virulent MDV variant belonging to an independent Chinese branch. To our knowledge, this is the first report to provide convincible evidence to identify a pathogenic MDV variant strain with a higher virulence than Md5 in China, which may have emerged and circulating in poultry farms in China for a long time and involved in the recent MD outbreaks.

Tibetan chickens are descendants of the ancestral red jungle fowl Gallus gallus. Very little is known about pathogens in Tibetan chickens living in the high-altitude environment. Here, we report for the first time the detection and isolation of avian leukosis virus from Tibetan chickens, with all the avian leukosis virus–positive samples belonging to subgroup J. Phylogenetic analysis of the sequence revealed these viruses were in a new branch compared with previous reports. The 3′-end of the pol gene in the new strains showed 8-amino acid deletion, with 2 strains displaying a large-scale deletion in the hr2 region of gp85 protein. Among all the strains, several mutations in the primer binding site leader sequence and untranslated region, which came from Rous-associated virus, were identified. It is interesting that some of these mutations may have contributed to the competitive advantages to these isolates as observed from their increased replication in vitro. These results indicated that the virus isolates from Tibetan chickens can have competitive advantage over the other strains circulating in the poultry population in future.

In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45–UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge.

Duck enteritis virus (DEV) and Pasteurella multocida, the causative agent of duck plague and fowl cholera, are acute contagious diseases and leading causes of morbidity and mortality in duck. The NHEJ-CRISPR/Cas9-mediated gene editing strategy, accompanied with the Cre–Lox system, have been employed in the present study to show that two new sites at UL55-LORF11 and UL44-44.5 loci in the genome of the attenuated Jansen strain of DEV can be used for the stable expression of the outer membrane protein H (ompH) gene of P. multocida that could be used as a bivalent vaccine candidate with the potential of protecting ducks simultaneously against major viral and bacterial pathogens. The two recombinant viruses, DEV-OmpH-V5-UL55-LORF11 and DEV-OmpH-V5-UL44-44.5, with the insertion of ompH-V5 gene at the UL55-LORF11 and UL44-44.5 loci respectively, showed similar growth kinetics and plaque size, compared to the wildtype virus, confirming that the insertion of the foreign gene into these did not have any detrimental effects on DEV. This is the first time the CRISPR/Cas9 system has been applied to insert a highly immunogenic gene from bacteria into the DEV genome rapidly and efficiently. This approach offers an efficient way to introduce other antigens into the DEV genome for multivalent vector.

MicroRNAs (miRNAs) have been demonstrated for their involvement in virus biology and pathogenesis, including functioning as key determinants of virally-induced cancers. As an important oncogenic α-herpesvirus affecting poultry health, Marek’s disease virus serotype 1 [Gallid alphaherpesvirus 2 (GaHV-2)] induces rapid-onset T-cell lymphomatous disease commonly referred to as Marek’s disease (MD), an excellent biological model for the study of virally-induced cancer in the natural hosts. Previously, we have demonstrated that GaHV-2-encoded miRNAs (especially those within the Meq-cluster) have the potential to act as critical regulators of multiple processes such as virus replication, latency, pathogenesis, and/or oncogenesis. In addition to miR-M4-5p (miR-155 homolog) and miR-M3-5p, we have recently found that miR-M2-5p possibly participate in inducing MD lymphomagenesis. Here, we report the identification of two tumor suppressors, the RNA-binding protein 24 (RBM24) and myogenic differentiation 1 (MYOD1), being two biological targets for miR-M2-5p. Our experiments revealed that as a critical miRNA, miR-M2-5p promotes cell proliferation via regulating the RBM24-mediated p63 overexpression and MYOD1-mediated IGF2 signaling and suppresses apoptosis by targeting the MYOD1-mediated Caspase-3 signaling pathway. Our data present a new strategy of a single viral miRNA exerting dual role to potentially participate in the virally-induced T-cell lymphomagenesis by simultaneously promoting the cell proliferation and suppressing apoptosis.

Deoxyribonucleic acid (DNA) damage response (DDR) is the fundamental cellular response for maintaining genomic integrity and suppressing tumorigenesis. The activation of ataxia telangiectasia-mutated (ATM) kinase is central to DNA double-strand break (DSB) for maintaining host-genome integrity in mammalian cells. Oncolytic Newcastle disease virus (NDV) can selectively replicate in tumor cells; however, its influence on the genome integrity of tumor cells is not well-elucidated. Here, we found that membrane fusion and NDV infection triggered DSBs in tumor cells. The late replication and membrane fusion of NDV mechanistically activated the ATM-mediated DSB pathway via the ATM-Chk2 axis, as evidenced by the hallmarks of DSBs, i.e., auto-phosphorylated ATM and phosphorylated H2AX and Chk2. Immunofluorescence data showed that multifaceted ATM-controlled phosphorylation markedly induced the formation of pan-nuclear punctum foci in response to NDV infection and F-HN co-expression. Specific drug-inhibitory experiments on ATM kinase activity further suggested that ATM-mediated DSBs facilitated NDV replication and membrane fusion. We confirmed that the Mre11-RAD50-NBS1 (MRN) complex sensed the DSB signal activation triggered by NDV infection and membrane fusion. The pharmacological inhibition of MRN activity also significantly inhibited intracellular and extracellular NDV replication and syncytia formation. Collectively, these data identified for the first time a direct link between the membrane fusion induced by virus infection and DDR pathways, thereby providing new insights into the efficient replication of oncolytic NDV in tumor cells.

Marek’s disease virus (MDV) is a member of alphaherpesviruses associated with Marek’s disease, a highly contagious neoplastic disease in chickens. The availability of the complete sequence of the viral genome allowed for the identification of major genes associated with pathogenicity using different techniques, such as bacterial artificial chromosome (BAC) mutagenesis and the recent powerful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based editing system. Thus far, most studies on MDV genome editing using the CRISPR/Cas9 system have focused on gene deletion. However, analysis of the expression and interactions of the viral proteins during virus replication in infected cells and tumor cells is also important for studying its role in MDV pathogenesis. The unavailability of antibodies against most of the MDV proteins has hindered the progress in such studies. This prompted us to develop pipelines to tag MDV genes as an alternative method for this purpose. Here we describe the application of CRISPR/Cas9 gene-editing approaches to tag the phosphoprotein 38 (pp38) gene of the MDV vaccine strain CVI988 with both V5 and green fluorescent protein (GFP). This rapid and efficient viral-gene-tagging technique can overcome the shortage of specific antibodies and speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.

Abstract Background The gp85 is the main envelope protein of avian leukosis subgroup J (ALV-J) involved in virus neutralization. Here, we mapped the epitope in ALV-J gp85 by ELISA using synthetic peptides and developed epitope based diagnostic methods for ALV-J infection. Results The results revealed that monoclonal antibody (mAb) JE9 recognized 83WDPQEL88 motif, which was highly conserved in gp85 among different ALV-J strains by homology analysis. Moreover, after evaluation with two hundred and forty sera samples obtained from different chicken farms, the epitope-based peptide ELISA had much higher sensitivity than commercial ELISA kit for antibody detection of ALV-J. Conclusions A novel B-cell epitope recognized by the mAb JE9 was identified. The developed peptide-ELISA based on this novel B-cell epitope could be useful in laboratory viral diagnosis, routine surveillance in chicken farms, and also in understanding the pathogenesis of ALV-J.

In the present study, we determined the in vitro characteristics and binding interactions of chicken PD-1 (chPD-1) and PD-L1 (chPD-L1) and developed a panel of specific monoclonal antibodies against the two proteins. ChPD-1 and chPD-L1 sequence identities and similarities were lower compared with those of humans and other mammalian species. Furthermore, in phylogenetic analysis, chPD-1 and chPD-L1 were grouped separately from the mammalian PD-1 and PD-L1 sequences. As in other species, chPD-1 and chPD-L1 sequences showed signal peptide, extracellular domain, a transmembrane domain and intracellular domain. Based on the three dimensional (3D) structural homology, chPD-1, and chPD-L1 were similar to 3D structures of mammalian PD-1 and PD-L1. Further, Ig V domain of chPD-1 and the Ig V and Ig C domains of chPD-L1 were highly conserved with the mammalian counterparts. In vitro binding interaction studies using Superparamagnetic Dynabeads® confirmed that recombinant soluble chPD-1/PD-L1 fusion proteins and surface chPD-1/PD-L1 proteins interacted with each other on COS cells. Two monoclonal antibodies specific against chPD-1 and five antibodies against chPD-L1 were developed and their specific binding characteristics confirmed by immunofluorescence staining and Western blotting.

Abstract Background Co-infection with avian leukosis virus subgroup J and reticuloendotheliosis virus induces synergistic pathogenic effects and increases mortality. However, the role of exosomal miRNAs in the molecular mechanism of the synergistic infection of the two viruses remains unknown. Results In this study, exosomal RNAs from CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time were analysed by Illumina RNA deep sequencing. A total of 54 (23 upregulated and 31 downregulated) and 16 (7 upregulated and 9 downregulated) miRNAs were identified by comparing co-infection with two viruses, single-infected ALV-J and REV, respectively. Moreover, five key miRNAs, including miR-184-3p, miR-146a-3p, miR-146a-5p, miR-3538 and miR-155, were validated in both exosomes and CEF cells by qRT-PCR. GO annotation and KEGG pathway analysis of the miRNA target genes showed that the five differentially expressed miRNAs participated in virus-vector interaction, oxidative phosphorylation, energy metabolism and cell growth. Conclusions We demonstrated that REV and ALV-J synergistically increased the accumulation of exosomal miRNAs, which sheds light on the synergistic molecular mechanism of ALV-J and REV.

Background/Aims: Newcastle disease virus (NDV) causes a highly devastating and contagious disease in poultry, which is mainly attributed to extensive tissue damages in the digestive, respiratory and nervous systems. However, nature and dynamics of NDV-induced oxidative stresses in the intestine of chickens remain elusive. Methods: In this study, we examined the magnitude of intestinal oxidative stress and histopathological changes caused by the virulent NDV infection, and explored the protective roles of vitamin E (vit. E) in ameliorating these pathological changes. For these purposes, chickens were divided into four groups namely i) non supplemented and non-challenged (negative control, CON); ii) no supplementation of vit. E but challenged with ZJ1 (positive control, NS+CHA); iii) vit. E supplementation at the dose of 50 IU/day/Kg body weight and ZJ1 challenge (VE50+CHA); and 4) vit. E supplementation at the dose of 100 IU/day/Kg body weight and ZJ1 challenge (VE100+CHA). In all groups, we analyzed concentrations of glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (T-AOC), and activity of glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) using biochemical methods. The virus loads were determined by quantitative RT-PCR and antibody titers by hemagglutination inhibition assays. We also examined the histopathological changes in the duodenal and jejunal mucosa at 3 and 5-day post infection (dpi) with NDV. Results: A significant elevation in the NO level was observed in NDV challenged chickens compared to the CON chickens at 2 dpi. The MDA contents were significantly increased whereas GSH was significantly decreased in NDV-challenged chickens compared to control. Furthermore, activities of GST, CAT, SOD, as well as the TOAC were markedly decreased in challenged chickens in comparison with control. Virus copy numbers were higher in NDV infected NS+CHA group compared to other groups. Severe histopathological changes including inflammation, degeneration and broken villi were observed in the intestine of NDV challenged chickens. However, all these malfunctions of antioxidant system and pathological changes in the intestine were partially or completely reversed by the vit. E supplementation. Conclusions: Our results suggest that NDV infection causes oxidative stress and histopathological changes in the duodenum and jejunum of chickens, which can be partially or fully ameliorated by supplementation of vit. E. Additionally, these findings suggest that oxidative stress contributes to the intestinal damages in NDV infected chickens. These findings will help to understand the pathogenesis of NDV and further investigation of therapeutic agents for control of Newcastle disease.

Abstract The intracellular actions of interferon (IFN)-regulated proteins, including IFN-induced proteins with tetratricopeptide repeats (IFITs), attribute a major component of the protective antiviral host defense. Here we applied genomics approaches to annotate the chicken IFIT locus and currently identified a single IFIT (chIFIT5) gene. The profound transcriptional level of this effector of innate immunity was mapped within its unique cis-acting elements. This highly virus- and IFN-responsive chIFIT5 protein interacted with negative sense viral RNA structures that carried a triphosphate group on its 5′ terminus (ppp-RNA). This interaction reduced the replication of RNA viruses in lentivirus-mediated IFIT5-stable chicken fibroblasts whereas CRISPR/Cas9-edited chIFIT5 gene knockout fibroblasts supported the replication of RNA viruses. Finally, we generated mosaic transgenic chicken embryos stably expressing chIFIT5 protein or knocked-down for endogenous chIFIT5 gene. Replication kinetics of RNA viruses in these transgenic chicken embryos demonstrated the antiviral potential of chIFIT5 in ovo. Taken together, these findings propose that IFIT5 specifically antagonize RNA viruses by sequestering viral nucleic acids in chickens, which are unique in innate immune sensing and responses to viruses of both poultry and human health significance.

Wnt/β-catenin signaling is a highly conserved pathway related to a variety of biological processes in different cells. The regulation of replication of various viruses by Wnt/β-catenin signaling pathway has been reported. However, the interaction between the Wnt/β-catenin pathway and avian leukosis virus is unknown. In the present study, we investigated the effect of modulating the Wnt/β-catenin pathway during avian leukosis virus subgroup J (ALV-J) infection. The activation of the Wnt/β-catenin pathway by GSK-3 inhibitor increased ALV-J mRNA, viral protein expression, and virus production in CEF cells. This increase was suppressed by iCRT14, one of the specific inhibitors of the Wnt/β-catenin signaling pathway. Moreover, treatment with iCRT14 reduced virus titer and viral gene expression significantly in CEF and LMH cells in a dose-dependent manner. Inhibition Wnt/β-catenin signaling pathway by knockdown of β-catenin reduced virus proliferation in CEF cells also. Collectively, these results suggested that the status of Wnt/β-catenin signaling pathway modulated ALV-J replication. These studies extend our understanding of the role of Wnt/β-catenin signaling pathway in ALV-J replication and make a new contribution to understanding the virus–host interactions of avian leukosis virus.

Marek’s disease (MD) is an immunosuppressive and highly contagious lymphoproliferative disease caused by Marek’s disease virus (MDV) in poultry. Lymphoblastoid cell lines (LCLs) generated ex vivo from MD lymphomas are considered excellent models to study virus-host molecular interactions. LCLs mostly have latently infected MDV genome, but many of them also have varying populations of lytically-infected cells, thus making them very suitable to examine the molecular events associated with the switch from latent to lytic infection. MDV-encoded phosphoprotein 38 (pp38) is readily detectable in lytically-infected LCLs and hence considered as a biomarker for lytic infection. Whilst previous studies have suggested that pp38 is essential for the early cytolytic infection of B-cells, its role in the switch from latent to lytic infection of LCLs is still unclear. pp24, another phosphorylated protein in the same protein complex, shares the same promoter and N-terminal 65 amino acids as pp38. In this study we employed CRISPR activation (CRISPRa) technology for targeted activation of pp38/pp24 in LCLs to investigate their role in inducing lytic infection. Our results show that enforced expression of pp38/pp24 through CRISPRa induces orchestrated upregulation of other MDV genes including ICP4, gB, Meq and pp14 as well as differential expression of host genes thereby facilitating lytic infection. Our results also show that pp38/pp24 expression induces the lytic switch through inhibiting apoptosis.

Abstract Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection.

Abstract Toll-like receptor-3 (TLR3), a member of the pathogen recognition receptor family, has been reported to activate immune response and to exhibit pro-apoptotic activity against some tumor cells. However it is unclear whether TLR3 has same function against chicken lymphoma. In this paper we investigated the effect of TLR3 activation on a Marek’s disease lymphoma-derived chicken cell line, MDCC-MSB1. The TLR3 agonist poly (I:C) activated TLR3 pathway and inhibited tumor cells proliferation through caspase-dependent apoptosis. Using pharmacological approaches, we found that an interferon-independent mechanism involving Toll-IL-1-receptor domain-containing adapter-inducing IFN-α (TRIF) and nuclear factor κB (NF-κB) causes the apoptosis of MDCC-MSB1 cells. This is the first report about the function of TLR3 in chicken T-cell lymphoma, especially in signal pathway. The mechanisms underlying TLR3-mediated apoptosis may contribute to the development of new drug to treat lymphomas and oncovirus infections.

Abstract Interferons (IFNs) are essential components of the host innate immune system and define first-line of defence against pathogens. In mammals, several type I IFNs are identified, however, only limited data is available on the repertoire of IFNs in avian species. Here we report the characterization of chicken IFN-κ (chIFN-κ) near the type I IFN locus on the sex-determining Z chromosome. Genetic, evolutionary and syntenic analyses indicate that chIFN-κ is a type I IFN with conserved genetic features and promoter binding sites. chIFN-κ regulated the IFN-stimulated response element signalling pathways and activated a panel of IFN-regulated genes, antiviral mediators and transcriptional regulators. Priming of chicken primary fibroblasts and tracheal organ cultures with chIFN-κ imparted cellular protections against viral infections both in vitro and ex vivo. To determine whether chIFN-κ defines the antiviral state in developing chicken embryos, we used replication-competent retroviral RCAS vector system to generate transgenic chicken embryos that constitutively and stably expressed chIFN-κ. We could demonstrate that chIFN-κ markedly inhibited the replication of avian RNA viruses in ovo. Collectively, these results shed the light on the repertoire of IFNs in avian species and provide functional data on the interaction of the chIFN-κ with RNA viruses of poultry and public health importance.

Viral diseases remain a major concern for animal health and global food production in modern agriculture. In chickens, avian leukosis virus subgroup J (ALV-J) represents an important pathogen that causes severe economic loss. Until now, no vaccine or antiviral drugs are available against ALV-J and strategies to combat this pathogen in commercial flocks are desperately needed. CRISPR/Cas9 targeted genome editing recently facilitated the generation of genetically modified chickens with a mutation of the chicken ALV-J receptor Na+/H+ exchanger type 1 (chNHE1). In this study, we provide evidence that this mutation protects a commercial chicken line (NHE1ΔW38) against the virulent ALV-J prototype strain HPRS-103. We demonstrate that replication of HPRS-103 is severely impaired in NHE1ΔW38 birds and that ALV-J-specific antigen is not detected in cloacal swabs at later time points. Consistently, infected NHE1ΔW38 chickens gained more weight compared to their non-transgenic counterparts (NHE1W38). Histopathology revealed that NHE1W38 chickens developed ALV-J typical pathology in various organs, while no pathological lesions were detected in NHE1ΔW38 chickens. Taken together, our data revealed that this mutation can render a commercial chicken line resistant to highly pathogenic ALV-J infection, which could aid in fighting this pathogen and improve animal health in the field.

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

The expression of accessory non-structural proteins V and W in Newcastle disease virus (NDV) infections depends on RNA editing. These proteins are derived from frameshifts of the sequence coding for the P protein via co-transcriptional insertion of one or two guanines in the mRNA. However, a larger number of guanines can be inserted with lower frequencies. We analysed data from deep RNA sequencing of samples from in vitro and in vivo NDV infections to uncover the patterns of mRNA editing in NDV. The distribution of insertions is well described by a simple Markov model of polymerase stuttering, providing strong quantitative confirmation of the molecular process hypothesised by Kolakofsky and collaborators three decades ago. Our results suggest that the probability that the NDV polymerase would stutter is about 0.45 initially, and 0.3 for further subsequent insertions. The latter probability is approximately independent of the number of previous insertions, the host cell, and viral strain. However, in LaSota infections, we also observe deviations from the predicted V/W ratio of about 3:1 according to this model, which could be attributed to deviations from this stuttering model or to further mechanisms downregulating the abundance of W protein.

The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek’s disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.

Marek’s disease is a major scourge challenging poultry health worldwide. It is caused by the highly contagious Marek’s disease virus (MDV), an alphaherpesvirus. Here, we showed that, similar to other members of its Herpesviridae family, MDV also presents a complex landscape of splicing events, most of which are uncharacterised and/or not annotated. Quite strikingly, and although the biological relevance of this fact is unknown, we found that a number of viral splicing isoforms are strain-specific, despite the close sequence similarity of the strains considered: very virulent RB-1B and vaccine CVI-988. We validated our findings by devising an assay that discriminated infections caused by the two strains in chicken embryonic fibroblasts on the basis of the presence of some RNA species. To our knowledge, this study is the first to accomplish such a result, emphasizing how relevant a comprehensive picture of the viral transcriptome is to fully understand viral pathogenesis.

Herpesvirus of turkeys (HVT), used originally as a vaccine against Marek’s disease (MD), has recently been shown to be a highly effective viral vector for generation of recombinant vaccines that deliver protective antigens of other avian pathogens. Until the recent launch of commercial HVT-vectored dual insert vaccines, most of the HVT-vectored vaccines in the market carry a single foreign gene and are usually developed with slow and less efficient conventional recombination methods. There is immense value in developing multivalent HVT-vectored vaccines capable of inducing simultaneous protection against multiple avian pathogens, particularly to overcome the interference between individual recombinant HVT vaccines. Here we demonstrate the use of a previously developed CRISPR/Cas9 gene editing protocol for the insertion of ILTV gD-gI and the H9N2 AIV hemagglutinin expression cassettes into the distinct locations of the recombinant HVT-IBDV VP2 viral genome, to generate the triple insert HVT-VP2-gDgI-HA recombinant vaccine. The insertion, protein expression, and stability of each insert were then evaluated by PCR, immunostaining and Western blot analyses. The successful generation of the first triple insert recombinant HVT vaccine with the potential for the simultaneous protection against three major avian viral diseases in addition to MD is a major innovation in vaccination-based control of major poultry diseases.

With total dependence on the host cell, several viruses have adopted strategies to modulate the host cellular environment, including the modulation of microRNA (miRNA) pathway through virus-encoded miRNAs. Several avian viruses, mostly herpesviruses, have been shown to encode a number of novel miRNAs. These include the highly oncogenic Marek’s disease virus-1 (26 miRNAs), avirulent Marek’s disease virus-2 (36 miRNAs), herpesvirus of turkeys (28 miRNAs), infectious laryngotracheitis virus (10 miRNAs), duck enteritis virus (33 miRNAs) and avian leukosis virus (2 miRNAs). Despite the closer antigenic and phylogenetic relationship among some of the herpesviruses, miRNAs encoded by different viruses showed no sequence conservation, although locations of some of the miRNAs were conserved within the repeat regions of the genomes. However, some of the virus-encoded miRNAs showed significant sequence homology with host miRNAs demonstrating their ability to serve as functional orthologs. For example, mdv1-miR-M4-5p, a functional ortholog of gga-miR-155, is critical for the oncogenicity of Marek’s disease virus. Additionally, we also describe the potential association of the recently described avian leukosis virus subgroup J encoded E (XSR) miRNA in the induction of myeloid tumors in certain genetically-distinct chicken lines. In this review, we describe the advances in our understanding on the role of virus-encoded miRNAs in avian diseases.

Subgroup J avian leukosis virus (ALV-J), an oncogenic retrovirus, causes hemangiomas and myeloid tumors in chickens. We previously showed that miR-125b is down-regulated in ALV-J-induced tumors. This study aimed to investigate the possible role of miR-125b in ALV-J-mediated infection and tumorigenesis. Knockdown of miR-125b expression in HP45 cells reduced, whereas over-expression induced late-stage apoptosis. Bioinformatics analysis and luciferase activity assays indicate that miR-125b targets Semaphorin 4D/CD100 (Sema4D) by binding the 3′-untranslated region of messenger RNA (mRNA). Up-regulation of miR-125b in the DF1 cell line suppressed Sema4D expression, whereas miR-125 down-regulation increased Sema4D expression levels. To uncover the function of Sema4D during ALV-J infection, animal infection experiments and in vitro assays were performed and show that Sema4D mRNA levels were up-regulated in ALV-J-infected tissues and cells. Finally, functional experiments show that miR-125 down-regulation and Sema4D over-expression inhibited apoptosis in HP45 cells. These results suggest that miR-125b and its target Sema4D might play an important role in the aggressive growth of HP45 cells induced by avian leukosis viruses (ALVs). These findings improve our understanding of the underlying mechanism of ALV-J infection and tumorigenesis.

Newcastle disease virus (NDV), an avian paramyxovirus, was shown to prefer to replicate in tumor cells instead of normal cells; however, this mechanism has not been fully elucidated. Exosomes play a crucial role in intercellular communication due to the bioactive substances they carry. Several studies have shown that exosomes are involved in virus infections. However, the effect that exosomes have on NDV-infected tumor cells is not known. In this study, we focus on the role of exosomes secreted by NDV-infected HeLa cells in promoting NDV replication. Three miRNA candidates (miR-1273f, miR-1184, and miR-198) embraced by exosomes were associated with enhancing NDV-induced cytopathic effects on HeLa cells. Furthermore, luciferase assays, RT-qPCR, and enzyme-linked immunosorbent assay (ELISA) all demonstrated that these miRNAs could suppress interferon (IFN)-β gene expression. Enhanced NDV replication in HeLa cells was identified by Western blot and plaque assays. Based on these results, we speculate that NDV employed exosomes entry into neighboring cells, which carry miRNAs, resulting in inhibition of the IFN pathway and promotion of viral infection. To our knowledge, this is the first report on the involvement of NDV-employed exosomes in tumor cells, and as such, it provides new insights into the development of anti-tumor therapies.

Marek’s disease virus (MDV), a lymphotropic α-herpesvirus associated with T-cell lymphomas in chickens, is an excellent model for herpesvirus biology and virus-induced oncogenesis. Marek’s disease (MD) is also one of the cancers against which a vaccine was first used. In the lymphomas and lymphoblastoid cell lines (LCLs) derived from them, MDV establishes latent infection with limited gene expression. Although LCLs are valuable for interrogating viral and host gene functions, molecular determinants associated with the maintenance of MDV latency and lytic switch remain largely unknown, mainly due to the lack of tools for in situ manipulation of the genomes in these cell lines. Here we describe the first application of CRISPR/Cas9 editing approach for precise editing of the viral gene phosphoprotein 38 (pp38), a biomarker for latent/lytic switch in MDV-transformed LCLs MDCC-MSB-1 (Marek’s disease cell line MSB-1) and MDCC-HP8. Contradictory to the previous reports suggesting that pp38 is involved in the maintenance of transformation of LCL MSB-1 cells, we show that pp38-deleted cells proliferated at a significant higher rate, suggesting that pp38 is dispensable for the transformed state of these cell lines. Application of CRISPR/Cas9-based gene editing of MDV-transformed cell lines in situ opens up further opportunities towards a better understanding of MDV pathogenesis and virus-host interactions.

MicroRNAs (miRNAs) are small noncoding RNAs that fine-tune the responses of the cell by modulating the cell transcriptome and gene expression. MicroRNA 155 (miR-155) is a conserved multifunctional miRNA involved in multiple roles including the modulation of the immune responses. When deregulated, miR-155 can also contribute to cancer as has been demonstrated in several human malignancies such as diffuse large B cell lymphoma, chronic lymphocytic leukemia, as well as in Epstein–Barr virus (EBV)-induced B cell transformation. Avian oncogenic viruses such as Marek’s disease virus (MDV), avian leukosis virus (ALV), and reticuloendotheliosis virus (REV) that account for more than 90% of cancers in avian species, also make use of the miR-155 pathway during oncogenesis. While oncogenic retroviruses, such as ALV, activate miR-155 by insertional activation, acutely transforming retroviruses use transduced oncogenes such as v-rel to upregulate miR-155 expression. MDV on the other hand, encodes a functional miR-155 ortholog mdv1-miR-M4, similar to the miR-155 ortholog kshv-miR-K11 present in Kaposi’s sarcoma-associated herpesvirus (KSHV). We have shown that mdv1-miR-M4 is critical for the induction of MDV-induced lymphomas further demonstrating the oncogenic potential of miR-155 pathway in cancers irrespective of the diverse etiology. In this review, we discuss on our current understanding of miR-155 function in virus-induced lymphomas focusing primarily on avian oncogenic viruses.

Marek’s disease virus (MDV) is a member of alphaherpesviruses associated with Marek’s disease, a highly contagious neoplastic disease in chickens. Complete sequencing of the viral genome and recombineering techniques using infectious bacterial artificial chromosome (BAC) clones of Marek’s disease virus genome have identified major genes that are associated with pathogenicity. Recent advances in CRISPR/Cas9-based gene editing have given opportunities for precise editing of the viral genome for identifying pathogenic determinants. Here we describe the application of CRISPR/Cas9 gene editing approaches to delete the Meq and pp38 genes from the CVI988 vaccine strain of MDV. This powerful technology will speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.

Newcastle disease (ND), characterized by visceral, respiratory, and neurological pathologies, causes heavy economic loss in the poultry industry around the globe. While significant advances have been made in effective diagnosis and vaccine development, molecular mechanisms of ND virus (NDV)-induced neuropathologies remain elusive. In this study, we report the magnitude of oxidative stress and histopathological changes induced by the virulent NDV (ZJ1 strain) and assess the impact of vitamin E in alleviating these pathologies. Comparative profiling of plasma and brains from mock and NDV-infected chicken demonstrated alterations in several oxidative stress makers such as nitric oxide, glutathione, malondialdehyde, total antioxidant capacity, glutathione S-transferase, superoxide dismutase, and catalases. While decreased levels of glutathione and total antioxidant capacity and increased concentrations of malondialdehyde and nitric oxide were observed in NDV-challenged birds at all time points, these alterations were eminent at latter time points (5 days post infection). Additionally, significant decreases in the activities of glutathione S-transferase, superoxide dismutase, and catalase were observed in the plasma and brains collected from NDV-infected chickens. Intriguingly, we observed that supplementation of vitamin E can significantly reduce the alteration of oxidative stress parameters. Under NDV infection, extensive histopathological alterations were observed in chicken brain including neural inflammation, capillary hyperemia, necrosis, and loss of prominent axons, which were reduced with the treatment of vitamin E. Taken together, our findings highlight that neurotropic NDV induces extensive tissue damage in the brain and alters plasma oxidative stress profiles. These findings also demonstrate that supplementing vitamin E ameliorates these pathologies in chickens and proposes its supplementation for NDV-induced stresses.

Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation.

Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek's disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a bona fide initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of 'closed loop' structure of mRNA.