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.

As one of the most important avian immunosuppressive and neoplastic diseases, Marek’s disease (MD), caused by oncogenic Marek’s disease virus (MDV), has caused huge economic losses worldwide over the past five decades. In recent years, MD outbreaks have occurred frequently in MD-vaccinated chicken flocks, but the key pathogenic determinants and influencing factors remain unclear. Herein, we analyzed the pathogenicity of seven newly isolated MDV strains from tumor-bearing chickens in China and found that all of them were pathogenic to chicken hosts, among which four MDV isolates, SDCW01, HNXZ05, HNSQ05 and HNSQ01, were considered to be hypervirulent MDV (HV-MDV) strains. At 73 days of the virus infection experiment, the cumulative incidences of MD were 100%, 93.3%, 90% and 100%, with mortalities of 83.3%, 73.3%, 60% and 86.7%, respectively, for the four viruses. The gross occurrences of tumors were 50%, 33.3%, 30% and 63.3%, respectively, accompanied by significant hepatosplenomegaly and serious atrophy of the immune organs. Furthermore, the immune protection effects of four commercial MD vaccines against SDCW01, CVI988, HVT, CVI988+HVT, and 814 were explored. Unexpectedly, during the 67 days of post-virus challenge, the protection indices (PIs) of these four MD vaccines were only 46.2%, 38.5%, 50%, and 28%, respectively, and the birds that received the monovalent CVI988 or HVT still developed tumors with cumulative incidences of 7.7% and 11.5%, respectively. To our knowledge, this is the first demonstration of the simultaneous comparison of the immune protection efficacy of multiple commercial MD vaccines with different vaccine strains. Our study revealed that the HV-MDV variants circulating in China could significantly break through the immune protection of the classical MD vaccines currently widely used. For future work, there is an urgent need to develop novel, more effective MD vaccines for tackling the new challenge of emerging HV-MDV strains or variants for the sustainable control of MD.

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.

Recent advancements in biotechnology have enabled the rapid identification and subsequent expression of pathogenic microbial major antigens that induce protective immune responses. However, subunit vaccines have not been successfully commercialized mainly due to the lack of sufficient levels of neutralizing antibodies (NAs). High levels of NA rely on the efficient recognition and cross-linking of multiple neutralizing epitopes with B-cell receptors (BCRs). Nanoparticles are able to display coupled antigenic arrays at high density and provide multiple binding molecular scenarios with BCRs. The high-resolution antigenic structure makes it possible to accurately display stable neutralizing epitopes. Therefore, the development of a nanovaccine that orientationally displays neutralizing epitopes is a feasible strategy. To address this hypothesis, the capsid (Cap) protein of porcine circovirus type 2 as model antigen was conjugated to gold nanoparticles (AuNPs) through direct reaction of the mercapto group of the unique cysteines with AuNPs, rendering Cap-AuNPs to have neutralizing epitopes on outer surface and an immunodominant epitope buried within the inner surface. In vitro studies showed that AuNPs promoted the phagocytosis of Cap protein and NA levels were significantly improved, meanwhile antibody levels against the immunodominant epitope was significantly reduced. In mouse studies, Cap-AuNP-immunized mice displayed a high production of interleukin (IL)-4, IL-10, and interferon-γ, suggesting that Cap-AuNPs can effectively activate CD4⁺ and CD8⁺ T cells and balance Th1 and Th2 cellular responses. This study presents a new vaccine design strategy based on antigen structure, where nanoparticles are coupled to antigens in well-ordered arrays and orientationally display neutralizing epitopes to enhance NA levels.

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.

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.