mRNA Splicing of UL44 and Secretion of Alphaherpesvirinae Glycoprotein C (gC) Is Conserved among the Mardiviruses.

Marek's disease (MD), caused by gallid alphaherpesvirus 2 (GaAHV2) or Marek's disease herpesvirus (MDV), is a devastating disease in chickens characterized by the development of lymphomas throughout the body. Vaccine strains used against MD include gallid alphaherpesvirus 3 (GaAHV3), a non-oncogenic chicken alphaherpesvirus homologous to MDV, and homologous meleagrid alphaherpesvirus 1 (MeAHV1) or turkey herpesvirus (HVT). Previous work has shown most of the MDV gC produced during in vitro passage is secreted into the media of infected cells although the predicted protein contains a transmembrane domain. We formerly identified two alternatively spliced gC mRNAs that are secreted during MDV replication in vitro, termed gC104 and gC145 based on the size of the intron removed for each UL44 (gC) transcript. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized GaAHV3 (strain 301B/1) and HVT also secrete gC due to mRNA splicing. To address this, we collected media from 301B/1- and HVT-infected cell cultures and used Western blot analyses and determined that both 301B/1 and HVT produced secreted gC. Next, we extracted RNAs from 301B/1- and HVT-infected cell cultures and chicken feather follicle epithelial (FFE) skin cells. RT-PCR analyses confirmed one splicing variant for 301B/1 gC (gC104) and two variants for HVT gC (gC104 and gC145). Interestingly, the splicing between all three viruses was remarkably conserved. Further analysis of predicted and validated mRNA splicing donor, branch point (BP), and acceptor sites suggested single nucleotide polymorphisms (SNPs) within the 301B/1 UL44 transcript sequence resulted in no gC145 being produced. However, modification of the 301B/1 gC145 donor, BP, and acceptor sites to the MDV UL44 sequences did not result in gC145 mRNA splice variant, suggesting mRNA splicing is more complex than originally hypothesized. In all, our results show that mRNA splicing of avian herpesviruses is conserved and this information may be important in developing the next generation of MD vaccines or therapies to block transmission.

RNA-seq analysis of chlorogenic acid intervention in duck embryo fibroblasts infected with duck plague virus.

INTRODUCTION: Chlorogenic acid, the primary active component in Chinese medicines like honeysuckle, exhibits anti-inflammatory and antiviral effects. It has been demonstrated that chlorogenic acid effectively prevents and treats Duck enteritis virus (DEV) infection. This study aims to further elucidate the mechanism by which chlorogenic acid prevents DEV infection. METHODS: Duck embryo fibroblast (DEF) cells were pre-treated with chlorogenic acid before being infected with DEV. Cell samples were collected at different time points for transcriptomic sequencing, while qPCR was used to detect the proliferation of DEV. Additionally, 30-day-old ducks were treated with chlorogenic acid, and their lymphoid organs were harvested for histopathological sections to observe pathological damage. The proliferation of DEV in the lymphoid organs was also detected using qPCR Based on the transcriptomic sequencing results, NF-κB1 gene was silenced by RNAi technology to analyze the effect of NF-κB1 gene on DEV proliferation. RESULTS: Compared to the viral infection group, DEF cells in the chlorogenic acid intervention group exhibited significantly reduced DEV load (P < 0.05). Transcriptomic sequencing results suggested that chlorogenic acid inhibited DEV proliferation in DEF cells by regulating NF-κB signaling pathway. The results of RNAi silencing suggested that in the three treatment groups, compared with the DEV experimental group, there was no significant difference in the effect of pre-transfection after transfection on DEV proliferation, while both the pre-transfection after transfection and the simultaneous transfection group showed significant inhibition on DEV proliferation Furthermore, compared to the virus infection group, ducks in the chlorogenic acid intervention group showed significantly decreased DEV load in their lymphoid organs (P < 0.05), along with alleviated pathological damage such as nuclear pyretosis and nuclear fragmentation. CONCLUSIONS: Chlorogenic acid effectively inhibits DEV proliferation in DEF and duck lymphatic organs, mitigates viral-induced pathological damage, and provides a theoretical basis for screening targeted drugs against DEV.

Molecular characterization of the meq oncogene of Marek's disease virus in vaccinated Brazilian poultry farms reveals selective pressure on prevalent strains.

Marek's disease virus (MDV) has become an increasingly virulent pathogen in the poultry industry despite vaccination efforts to control it. Brazil has experienced a significant rise of Marek's disease (MD) outbreaks in recent years. Our study aimed to analyze the complete meq gene sequences to understand the molecular epidemiological basis of MD outbreaks in Brazilian vaccinated layer farms. We detected a high incidence rate of visceral MD (67.74%) and multiple circulating MDV strains. The most prevalent and geographically widespread genotype presented several clinical and molecular characteristics of a highly virulent strain and evolving under positive selective pressure. Phylogenetic and phylogeographic analysis revealed a closer relationship with strains from the USA and Japan. This study sheds light on the circulation of MDV strains capable of infecting vaccinated birds. We emphasize the urgency of adopting preventive measures to manage MDV outbreaks threatening the poultry farming industry.

Tracing the origin of virulence.

Microbial genomes from ancient chickens uncover the drivers of pathogenicity.

Phenotypic Characterization of Recombinant Marek's Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence.

Marek's disease (MD) is a highly infectious lymphoproliferative disease in chickens with a significant economic impact. Mardivirus gallidalpha 2, also known as Marek's disease virus (MDV), is the causative pathogen and has been categorized based on its virulence rank into four pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+). A prior comparative genomics study suggested that several single-nucleotide polymorphisms (SNPs) and genes in the MDV genome are associated with virulence, including nonsynonymous (ns) SNPs in eight open reading frames (ORF): UL22, UL36, UL37, UL41, UL43, R-LORF8, R-LORF7, and ICP4. To validate the contribution of these nsSNPs to virulence, the vv+MDV strain 686 genome was modified by replacing nucleotides with those observed in the vMDV strains. Pathogenicity studies indicated that these substitutions reduced the MD incidence and increased the survival of challenged birds. Furthermore, using the best-fit pathotyping method to rank the virulence, the modified vv+MDV 686 viruses resulted in a pathotype similar to the vvMDV Md5 strain. Thus, these results support our hypothesis that SNPs in one or more of these ORFs are associated with virulence but, as a group, are not sufficient to result in a vMDV pathotype, suggesting that there are additional variants in the MDV genome associated with virulence, which is not surprising given this complex phenotype and our previous finding of additional variants and SNPs associated with virulence.

N-Linked Glycosylation and Expression of Duck Plague Virus pUL10 Promoted by pUL49.5.

Duck plague virus (DPV) is a member of the alphaherpesvirus subfamily, and its genome encodes a conserved envelope protein, protein UL10 (pUL10). pUL10 plays complex roles in viral fusion, assembly, cell-to-cell spread, and immune evasion, which are closely related to its protein characteristics and partners. Few studies have been conducted on DPV pUL10. In this study, we identified the characteristics of pUL10, such as the type of glycosylation modification and subcellular localization. The characteristic differences in pUL10 in transfection and infection suggest that there are other viral proteins that participate in pUL10 modification and localization. Therefore, pUL49.5, the interaction partner of pUL10, was explored. We found that pUL10 interacts with pUL49.5 during transfection and infection. Their interaction entailed multiple interaction sites, including noncovalent forces in the pUL49.5 N-terminal domains and C-terminal domains and a covalent disulfide bond between two conserved cysteines. pUL49.5 promoted pUL10 expression and mature N-linked glycosylation modification. Moreover, deletion of UL49.5 in DPV caused the molecular mass of pUL10 to decrease by approximately3 to 10 kDa, which suggested that pUL49.5 was the main factor affecting the N-linked glycosylation of DPV pUL10 during infection. This study provides a basis for future exploration of the effect of pUL10 glycosylation on virus proliferation. IMPORTANCE Duck plague is a disease with high morbidity and mortality rates, and it causes great losses for the duck breeding industry. Duck plague virus (DPV) is the causative agent of duck plague, and DPV UL10 protein (pUL10) is a homolog of glycoprotein M (gM), which is conserved in herpesviruses. pUL10 plays complex roles in viral fusion, assembly, cell-to-cell spread, and immune evasion, which are closely related to its protein characteristics and partners. In this study, we systematically explored whether pUL49.5 (a partner of pUL10) plays roles in the localization, modification, and expression of pUL10.

Deletion of Double Copies of the US1 Gene Reduces the Infectivity of Recombinant Duck Plague Virus In Vitro and In Vivo.

Duck plague caused by duck plague virus (DPV) is one of the main diseases that seriously endangers the production of waterfowl. DPV possesses a large genome consisting of 78 open reading frames (ORFs), and understanding the function and mechanism of each encoded protein in viral replication and pathogenesis is the key to controlling duck plague outbreaks. US1 is one of the two genes located in the repeat regions of the DPV genome, but the function of its encoded protein in DPV replication and pathogenesis remains unclear. Previous studies found that the US1 gene or its homologs exist in almost all alphaherpesviruses, but the loci, functions, and pathogenesis of their encoded proteins vary among different viruses. Here, we aimed to define the roles of US1 genes in DPV infection and pathogenesis by generating a double US1 gene deletion mutant and its revertant without any mini-F cassette retention. In vitro and in vivo studies found that deletion of both copies of the US1 gene significantly impaired the replication, gene expression, and virulence of DPV, which could represent a potential candidate vaccine strain for the prevention of duck plague. IMPORTANCE Duck plague virus contains nearly 80 genes, but the functions and mechanisms of most of the genes have not yet been elucidated, including those of the newly identified immediate early gene US1. Here, we found that US1 deletion reduces viral gene expression, replication, and virus production both in vitro and in vivo. This insight defines a fundamental role of the US1 gene in DPV infection and indicates its involvement in DPV transcription. These results provide clues for the study of the pathogenesis of the US1 gene and the development of attenuated vaccines targeting this gene.

V5 and GFP Tagging of Viral Gene pp38 of Marek's Disease Vaccine Strain CVI988 Using CRISPR/Cas9 Editing.

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.

Molecular characterization of duck plague virus from selected Haor areas of Bangladesh.

Background: Duck viral enteritis, commonly known as duck plague (DP), is an acute and contagious fatal disease in ducks, geese, and swans caused by the DP virus (DPV). It poses a serious threat to the growth of duck farming in the Haor (wetland) areas of Bangladesh. Aim: This study aimed to detect the circulating DPV by molecular characterization, followed by phylogenetic analysis, targeting the UL30 gene in infected ducks from five Haor districts in Bangladesh and to observe the variation in the genome sequence between the field virus and vaccine strain of DPV. Methods: A total of 150 samples (liver, 50; intestine, 50; and oropharyngeal tissue, 50) were collected from DP-suspected sick/dead ducks from 50 affected farms in Kishoreganj, Netrokona, B. Baria, Habiganj, and Sunamganj districts in Bangladesh. For the identification of DPV in collected samples, polymerase chain reaction (PCR) was utilized. Nucleotide sequences of the amplified UL30 gene were compared with those of other DPV strains available in GenBank. Results: Of the 150 samples, 90 (60%) were found to be positive for DPV, as confirmed by PCR. Organ-wise prevalence was higher in the liver (72%), followed by the intestine (64%) and oropharyngeal tissue (44%). Regarding areas, the highest and lowest prevalence in the liver and intestine was observed in Habiganj and B. Baria, respectively, whereas the highest and lowest prevalence in the oropharyngeal tissue was observed in B. Baria and Habiganj, respectively. Two isolates, BAU/KA/DPV(B1)/2014 from Kishoreganj and BAU/KA/DPV(B4)/2014 from Sunamganj were sequenced, and phylogenetic analysis revealed that these isolates are evolutionarily closely related to Chinese isolates of DPV. Additionally, the isolates of DPV BAU/KA/DPV(B1)/2014 and BAU/KA/DPV(B4)/2014 showed the highest (98%) similarity to each other. The nucleotide sequence of the isolate BAU/KA/DPV(B1)/2014 exhibited higher nucleotide variability (246 nucleotides) than that of the vaccine strain (accession no. EU082088), which may affect protein function and additional drug sensitivity. Conclusion: Based on the findings of the molecular study, it can be assumed that the Bangladeshi isolates and all Chinese isolates of DPV may have a common ancestry.

The intracellular domain of duck plague virus glycoprotein E affects UL11 protein incorporation into viral particles.

Studies have shown that proteins in the tegument (located between the viral capsid and envelope layer) play critical roles in the assembly and budding of herpesviruses. The UL11 protein of herpesviruses is important in the process of virus particle cell entry, release, assembly and secondary envelopment. Herpesvirus glycoprotein E (gE) is involved in syncytia formation, transmission between cells and nerve invasion. In herpes simplex virus, UL11 has been shown to interact with gE. However, little is known about the relationship of duck plague virus (DPV) pUL11 and gE. In this study, we constructed DPV cytoplasmic domain (CT)-gE, and extracellular domain (ET)-gE deletion mutants, pCMV-gE, CT-gE, and ET-gE and UL11 recombinant plasmids. We found that pUL11 can interact and colocalize with gE, CT-gE and ET-gE. Together, these results highlight an important role for UL11 in the function of gE, and may also have important implications for the role of pUL11 and gE.

UL28 and UL33 homologs of Marek's disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells.

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.

Two nuclear localization signals regulate intracellular localization of the duck enteritis virus UL13 protein.

Duck enteritis virus (DEV) multifunctional tegument protein UL13 is predicted to be a conserved herpesvirus protein kinase; however, little is known about its subcellular localization signal. In this study, through transfection of 2 predicted nuclear signals of DEV UL13 fused to enhanced green fluorescent protein, 2 bipartite nuclear localization signals (NLS) were identified. We found that ivermectin blocked the NLS-mediated nuclear import of DEV UL13, showing that the nuclear localization signal of DEV UL13 is a classical importin α- and ß-dependent process. We constructed a DEV UL13 mutant strain in which the NLS of DEV UL13 was deleted to explore whether deletion of the NLS affects viral replication. Amino acids 4 to 7 and 90 to 96 were predicted to be NLSs, further proving that nuclear import occurs via a classical importin α- and ß-dependent process. We also found that the NLS of pUL13 had no effect on DEV replication in cell culture. Our study enhances the understanding of DEV pUL13. Taken together, these results provide significant information regarding the biological function of pUL13 during DEV infection.

Genetic characterization of a Marek's disease virus strain isolated in Japan.

BACKGROUND: Marek's disease virus (MDV) causes malignant lymphomas in chickens (Marek's disease, MD). MD is currently controlled by vaccination; however, MDV strains have a tendency to develop increased virulence. Distinct diversity and point mutations are present in the Meq proteins, the oncoproteins of MDV, suggesting that changes in protein function induced by amino acid substitutions might affect MDV virulence. We previously reported that recent MDV isolates in Japan display distinct mutations in Meq proteins from those observed in traditional MDV isolates in Japan, but similar to those in MDV strains isolated from other countries. METHODS: To further investigate the genetic characteristics in Japanese field strains, we sequenced the whole genome of an MDV strain that was successfully isolated from a chicken with MD in Japan. A phylogenetic analysis of the meq gene was also performed. RESULTS: Phylogenetic analysis revealed that the Meq proteins in most of the Japanese isolates were similar to those of Chinese and European strains, and the genomic sequence of the Japanese strain was classified into the Eurasian cluster. Comparison of coding region sequences among the Japanese strain and MDV strains from other countries revealed that the genetic characteristics of the Japanese strain were similar to those of Chinese and European strains. CONCLUSIONS: The MDV strains distributed in Asian and European countries including Japan seem to be genetically closer to each other than to MDV strains from North America. These findings indicate that the genetic diversities of MDV strains that emerged may have been dependent on the different vaccination-based control approaches.

Research Note: Duck plague virus glycoprotein I influences cell-cell spread and final envelope acquisition.

To determine the role of glycoprotein I (gI) in duck plague virus (DPV), a gI-deleted mutant (BAC-CHv-ΔgI) and a gI-revertant virus (BAC-CHv-ΔgI Rev) were constructed by using a markerless two-step Red recombination system implemented on the DPV genome cloned into a bacterial artificial chromosome (BAC). Mutants were characterized on duck embryo fibroblast (DEF) cells compared with wild-type virus. BAC-CHv-ΔgI produced viral plaques on DEF cells that were on average approximately 57.2% smaller than those produced by BAC-CHv-ΔgI Rev and wild-type virus. Electron microscopy confirmed that deleting of gI resulted in nucleocapsids accumulated around the cytoplasm vesicles and few of them could complete the final envelopment process. These results clearly indicated that DPV gI plays significant roles in viral cell-cell spread and viral final envelopment process.

Construction of the recombinant duck enteritis virus delivering capsid protein VP0 of the duck hepatitis A virus.

Duck hepatitis A virus type 1 (DHAV-1) disease causes significant economic losses to the duck industry. Duck enteritis virus (DEV) is frequently used as a viral vector for aquatic poultry vaccination, but no recombinan DEV expressing DHAV-1 VP0 has been developed. In this study, we established a system for rescuing the DEV C-KCE vaccine strain by transfecting cells with six fosmid DNAs. We generated a recombinant virus (rDEV-ul41VP0) by inserting the VP0 gene of DHAV-1 into the ul41 region in the DEV C-KCE genome. DHAV-1 VP0 was stably expressed in the rDEV-ul41VP0 infected cells, but did not affect the replication properties of DEV C-KCE in cells. Duck experiments showed that rDEV-ul41VP0 could provided full protection against the lethal DEV Chinese standard challenge (DEV CSC) and conferred 70% protection against DHAV-1 161/79 at 3 days postvaccination. These results indicate that rDEV-ul41VP0 rapidly induces protection against DEV CSC and DHAV-1 in ducks, and can be served as a bivalent vaccine against DEV and DHAV-1.

Role of cholesterol in anatid herpesvirus 1 infections in vitro.

Cholesterol is an essential constituent of the cell membrane that modulates several physiological events, including virus entry into the host. Duck virus enteritis (DVE) is a contagious and lethal infection that attacks several species of waterfowl. Anatid herpesvirus 1 (AnHV-1) is the causative agent of duck viral enteritis and classified under subfamily Alphaherpesvirinae. In this study, the effect of cholesterol depletion in both host cell membrane and viral envelope on the infectivity of AnHV-1 was explored. Cholesterol depletion of chicken embryo fibroblast cells (DF-1) by methyl-ß-cyclodextrin (MßCD) inhibited the infectivity of AnHV-1. This inhibitory effect was moderately reversed by the exogenous replenishment of cholesterol in the cells. Furthermore, the inhibition of endogenous cholesterol synthesis by a statin drug also inhibited the infectivity of AnHV-1. Presumably, the removal of cholesterol from AnHV-1 envelope might be disrupting the viral envelope resulting in its diminished infectivity. The presence of a relatively hydrophobic cavity in MßCD can be used to extract cholesterol from the cell membrane. Loss of infectivity of the virus might be due to the effects of MßCD mediated cholesterol depletion from the cell membrane. The results implicate that the cell membrane cholesterol is vital for the infectivity of AnHV-1 in DF-1 cells, and its depletion from virion curtails the infectivity by destabilizing the envelope.

The bZIP Proteins of Oncogenic Viruses.

Basic leucine zipper (bZIP) transcription factors (TFs) govern diverse cellular processes and cell fate decisions. The hallmark of the leucine zipper domain is the heptad repeat, with leucine residues at every seventh position in the domain. These leucine residues enable homo- and heterodimerization between ZIP domain α-helices, generating coiled-coil structures that stabilize interactions between adjacent DNA-binding domains and target DNA substrates. Several cancer-causing viruses encode viral bZIP TFs, including human T-cell leukemia virus (HTLV), hepatitis C virus (HCV) and the herpesviruses Marek's disease virus (MDV), Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). Here, we provide a comprehensive review of these viral bZIP TFs and their impact on viral replication, host cell responses and cell fate.

Abrogation of Marek's disease virus replication using CRISPR/Cas9.

Marek's disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen.

Complete genome sequence of an isolate of duck enteritis virus from China.

Here, we present the complete genomic sequence of duck enteritis virus (DEV) strain SD, isolated in China in 2012. The virus was virulent in experimentally infected 2-month-old ducks. The DEV SD genome is 160,945 base pairs (bp) in length. The viral genome sequence, when compared to that of strain DEV CSC, which was isolated in 1962, showed three discontinuous deletions of 101 bp, 48 bp and 417 bp within the inverted repeats. A comparison of the amino acid (aa) sequences of all ORFs of the CSC and SD isolates demonstrated an11-aa deletion, two single-aa deletions, and one single-aa deletion in LORF3, UL47, UL4, respectively. Moreover, 38 single aa variations were also detected in 24 different ORFs. These results will further advance our understanding of the genetic variations involved in evolution.

The Pivotal Roles of US3 Protein in Cell-to-Cell Spread and Virion Nuclear Egress of Duck Plague Virus.

The duck plague virus (DPV) US3 protein, a homolog of the herpes simplex virus-1 (HSV-1) US3 protein that is reported to be critical for viral replication, has been minimally studied. Therefore, to investigate the function of the DPV US3 protein, we used scarless Red recombination technology based on an infectious bacterial artificial chromosome (BAC) containing the DPV Chinese virulent strain (CHv) genome and successfully constructed and rescued a US3-deleted mutant and the corresponding revertant virus (BAC-CHv-ΔUS3 and BAC-CHv-ΔUS3R, respectively). For viral growth characteristics, compared to the parental and revertant viruses, the US3-deleted mutant showed an approximately 100-fold reduction in viral titers but no significant reduction in genome copies, indicating that the US3-deleted mutant exhibited decreased viral replication but not decreased viral DNA generation. In addition, the US3-deleted mutant formed viral plaques that were 33% smaller on average than those formed by the parental and revertant viruses, demonstrating that US3 protein affected the viral cell-to-cell spread of DPV. Finally, the results of electron microscopy showed that the deletion of US3 resulted in a large number of virions accumulating in the nucleus and perinuclear space, thus blocking virion nuclear egress. In this study, we found that the DPV US3 protein played pivotal roles in viral replication by promoting viral cell-to-cell spread and virion nuclear egress, which may provide some references for research on the function of the DPV US3 protein.