Advancing vaccine development: Evaluation of a mannose-modified lipid nanoparticle-based candidate for African swine fever p30 mRNA vaccine eliciting robust immune response in mice.

The African swine fever virus (ASFV) continues to pose significant economic and pandemic risks. Consequently, discovering new, efficient vaccines is crucial. Messenger RNA (mRNA) vaccines have emerged as promising candidates, providing minimal risk of insertional mutagenesis, high safety profiles, effectiveness, rapid scalability in production, and cost-effectiveness. In this study, we have developed an ASF p30 mRNA vaccine candidate (mRNA/Man-LNP) employing mannose-modified lipid nanoparticles (LNPs). The mRNA/Man-LNP exhibited effective antigen presentation and facilitated dendritic cells (DCs) maturation. Notably, it elicited strong IgG titers and activated CD4+ and CD8+ T-cells in immunized mice, all while adhering to stringent biosafety standards. This investigation demonstrates that mRNA/Man-LNP can trigger both humoral and cellular immune responses, suggesting its potential as a potent and promising vaccine candidate for controlling African swine fever (ASF).

A candidate nanoparticle vaccine comprised of multiple epitopes of the African swine fever virus elicits a robust immune response.

The African swine fever (ASF) pandemics pose a significant threat to the global swine industry, and the development of safe and effective vaccines is a daunting but necessary challenge. The level and persistence of immunity are very important for the effectiveness of the vaccine. Targeting antigens to antigen presenting cells (APCs) can greatly enhance immunogenicity. In this study, we developed a self-assembled nano-ASFV vaccine candidate (NanoFVax) targeting DCs, by covalently coupling the self-assembled 24-mer ferritin with the dominant B and T cell epitopes of the highly immunogenic ASFV antigen (p72, CD2v, pB602L and p30) and fused with the chemokine receptor XCL1 (a DC targeting molecule) through the SpyTag/SpyCatcher protein ligase system. Compared to monomeric protein, the nanoparticle vaccines can induce a more robust T-cell response, and the high-level antibody response against ASFV can last for more than 231 days. Therefore, the NanoFVax is a novel and promising vaccine candidate for ASFV.

LORF9 of Marek's disease virus is involved in the early cytolytic replication of B lymphocytes and can act as a target for gene deletion vaccine development.

IMPORTANCE: Marek's disease virus (MDV) is a highly infectious and oncogenic virus that can induce severe T cell lymphomas in chickens. MDV encodes more than 100 genes, most of which have unknown functions. This work indicated that the LORF9 gene is necessary for MDV early cytolytic replication in B lymphocytes. In addition, we have found that the LORF9 deletion mutant has a comparative immunological protective effect with CVI988/Rispens vaccine strain against very virulent MDV challenge. This is a significant discovery that LORF9 can be exploited as a possible target for the development of an MDV gene deletion vaccine.

Antagonistic effects of N-acetylcysteine on lead-induced apoptosis and oxidative stress in chicken embryo fibroblast cells.

Lead (Pb) is a heavy metal that can have harmful effects on the environment, which has severe cytotoxicity in many animal tissues. N-acetylcysteine (NAC) has antioxidant activity, reducing lead-induced oxidative stress and apoptosis, but its role in chicken cells is unknown. The current study explored the antagonistic effect of NAC on lead-induced apoptosis and oxidative stress in chicken embryo fibroblast (CEF). In this study, CEF was used as a model to measure the cytotoxic effects of lead nitrate at different concentrations, demonstrating a dose-dependent effect on CEF activity. Employing inverted microscopy, the investigation of morphological alterations in CEF cells was conducted. Fluorescence staining methodology enabled the assessment of reactive oxygen species (ROS) levels within CEF cells. Moreover, an enzyme-linked immunosorbent assay was utilized to detect the presence of oxidative damage indicators encompassing superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activity, malondialdehyde (MDA) content, and total antioxidant capacity (T-AOC) within CEF cells. Furthermore, the determination of the apoptosis rate of CEF cells was accomplished through the utilization of the Hoechst 33258 staining method in combination with the Annexin V-FITC dual staining method. By using RT-qPCR for detection, lead treatment increased expression of pro-apoptotic genes, caspase-3, and caspase-9, and reduced expression of anti-apoptotic genes, Bcl-2, and BI-1. Reduced antioxidant capacity was shown by increased ROS and MDA levels in CEF cells after lead treatment. The results showed that NAC inhibited the expression of caspase-3 and caspase-9 in lead-treated CEF cells, while NAC had a certain inhibitory effect on the relative expression of Bcl-2 and BI-1 mRNA in lead-induced CEF cells. NAC significantly reduced lead-induced oxidative damage and apoptosis. Overall, our results demonstrate a novel protective effect of NAC against lead-induced injury in chicken cells, providing a theoretical basis for future investigations of drugs that are effective in preventing lead poisoning in animals.

Establishment and characterization of a novel indirect ELISA method based on ASFV antigenic epitope-associated recombinant protein.

African Swine Fever (ASF) is an acute and highly lethal disease in pigs caused by African Swine Fever Virus (ASFV). Viral proteins have been commonly used as antigenic targets for the development of ASF diagnostic methods. However, the prokaryotic expression of viral proteins has deficiencies such as instability, insolubility, and high cost in eukaryotic situations. This study screened and verified ASFV-encoded p72, p54, and p30 protein antigenic epitopes. Subsequently, a novel antigenic epitope-associated recombinant protein was designed based on an ideal structural protein and expressed in Escherichia coli (E. coli). Western blot analysis indicated that the recombinant protein could specifically react with the monoclonal antibody (mAb) of p72 and polyclonal antibodies of p54 and p30, respectively. Next, an ASF indirect ELISA (iELISA) method was established based on the recombinant protein, which has no specific reaction with sera of other important pig viral diseases. Meanwhile, it shows a sensitivity to detecting dilutions of ASF-positive reference serum up to 1:6400. The clinical sample detection results showed a high coincidence rate of 98 % with a commercial competition ELISA kit. In conclusion, we established a novel specific, and sensitive ASF serologic detection method that opens new avenues for ASF serodiagnostic method development.

Establishment of a Dual-Antigen Indirect ELISA Based on p30 and pB602L to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF) is an acute, virulent, and highly fatal infectious disease caused by the African swine fever virus (ASFV). There is no effective vaccine or diagnostic method to prevent and control this disease currently, which highlights the significance of ASF early detection. In this study, we chose an early antigen and a late-expressed antigen to co-detect the target antibody, which not only helps in early detection but also improves accuracy and sensitivity. CP204L and B602L were successfully expressed as soluble proteins in an Escherichia coli vector system. By optimizing various conditions, a dual-antigen indirect ELISA for ASFV antibodies was established. The assay was non-cross-reactive with antibodies against the porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine circovirus type 2, and pseudorabies virus. The maximum serum dilution for detection of ASFV-positive sera was 1:1600. The intra-batch reproducibility coefficient of variation was <5% and the inter-batch reproducibility coefficient of variation was <10%. Compared with commercial kits, the dual-antigen indirect ELISA had good detection performance. In conclusion, we established a detection method with low cost, streamlined production process, and fewer instruments. It provides a new method for the serological diagnosis of ASF.

An Intracellular Epitope of ASFV CD2v Protein Elicits Humoral and Cellular Immune Responses.

The African swine fever virus (ASFV) causes high mortality in domestic pigs. ASFV encodes an important protein target for subunit vaccine development, CD2v, but its most effective immunological regions are not known. Herein, we generated a monoclonal antibody (mAb) named IF3 by immunizing mice against the intracellular region of the CD2v protein (CD2v-IR). 1F3 specifically recognized CD2v, which is expressed transiently in transfected Sf9 cells and also in inactivated ASFV-infected porcine alveolar macrophage (PAM) cells. The epitope recognized by 1F3 is 264EPSPREP270, which is highly conserved in ASFV genotypes. Immunization of mice with this epitope elicited an increased IgG response, including IgG1 and IgG2a subtypes, and also increased CD8+ T cells and cytokine expression. Overall, these results indicate that this epitope induces both humoral and cellular immune responses that may be used for ASFV-related subunit vaccine design and development.

Identification of a Linear B Cell Epitope on p54 of African Swine Fever Virus Using Nanobodies as a Novel Tool.

African swine fever (ASF) has received great attention from the swine industry due to the pandemic and the lack of vaccines or effective treatments. In the present study, 13 African swine fever virus (ASFV) p54-specific nanobodies (Nbs) were successfully screened based on Bactrian camel immunization of p54 protein and phage display technology, and their reactivity with the p54 C-terminal domain (p54-CTD) was determined; however, only Nb8-horseradish peroxidase (Nb8-HRP) exhibited the best reactivity. Immunoperoxidase monolayer assay (IPMA) and immunofluorescence assay (IFA) results indicated that Nb8-HRP specifically reacted with ASFV-infected cells. Then, the possible epitopes of p54 were identified using Nb8-HRP. The results showed that Nb8-HRP could recognize p54-CTD truncated mutant p54-T1. Then, 6 overlapping peptides covering p54-T1 were synthesized to determine the possible epitopes. Dot blot and peptide-based enzyme-linked immunosorbent assay (ELISA) results suggested that one novel minimal linear B cell epitope, 76QQWVEV81, which had never been reported before, was identified. Alanine-scanning mutagenesis revealed that 76QQWV79 was the core binding site for Nb8. Epitope 76QQWVEV81 was highly conserved among genotype II ASFV strains and could react with inactivated ASFV antibody-positive serum from naturally infected pigs, indicating that it was a natural linear B cell epitope. These findings provide valuable insights for vaccine design and p54 as an effective diagnostic tool. IMPORTANCE The ASFV p54 protein plays an important role in inducing neutralization antibodies in vivo after viral infection and is often used as a candidate protein for subunit vaccine development. The full understanding of the p54 protein epitope provides a sufficient theoretical basis for p54 as a vaccine candidate protein. The present study uses a p54-specific nanobody as a probe to identify a highly conserved antigenic epitope, 76QQWVEV81, among different ASFV strains, and it can induce humoral immune responses in pigs. This is the first report using virus-specific nanobodies as a tool to identify some special epitopes that cannot be recognized by conventional monoclonal antibodies. This study opens up nanobodies as a new tool for identifying epitopes and also provides a theoretical basis for understanding p54-induced neutralizing antibodies.

Identification of a linear B-cell epitope on the African swine fever virus CD2v protein.

African swine fever virus (ASFV) poses a serious threat to domestic pigs and wild boars, which is responsible for substantial production and economic losses. A dominant ASFV specific linear B cell epitope that reacted with the convalescent serum was explored and identified with the help of immune informatics techniques. It is essential in understanding the host immunity and in developing diagnostic technical guidelines and vaccine design. The confirmation of dominant epitopes with a positive serological matrix is feasible. To improve the immunogenicity of the epitope, we designed the dominant epitope of CD2v in the form of 2 branch Multiple-Antigen peptide (MAPs-2), CD2v-MAPs-2. Notably, CD2v peptide can be taken up by dendritic cells (DCs) to activate T lymphocytes and induce highly effective valence antibodies in BALB/c mice. The specific CD8+ T cell response were observed. The dominant epitope peptide identified in this study was able to effectively activate humoral and cellular immunity in mice model.

Infection phase-dependent dynamics of the viral and host N6-methyladenosine epitranscriptome in the lifecycle of an oncogenic virus in vivo.

Dynamic alteration of the epitranscriptome exerts regulatory effects on the lifecycle of oncogenic viruses in vitro. However, little is known about these effects in vivo because of the general lack of suitable animal infection models of these viruses. Using a model of rapid-onset Marek's disease lymphoma in chickens, we investigated changes in viral and host messenger RNA (mRNA) N6-methyladenosine (m6 A) modification during Marek's disease virus (MDV) infection in vivo. We found that the expression of major epitranscriptomic proteins varies among viral infection phases, reprogramming both the viral and the host epitranscriptomes. Specifically, the methyltransferase-like 3 (METTL3)/14 complex was suppressed during the lytic and reactivation phases of the MDV lifecycle, whereas its expression was increased during the latent phase and in MDV-induced tumors. METTL3/14 overexpression inhibits, whereas METTL3/14 knockdown enhances, MDV gene expression and replication. These findings reveal the dynamic features of the mRNA m6 A modification program during viral replication in vivo, especially in relation to key pathways involved in tumorigenesis.

Fully Attenuated meq and pp38 Double Gene Deletion Mutant Virus Confers Superior Immunological Protection against Highly Virulent Marek's Disease Virus Infection.

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.

HRP-conjugated-nanobody-based cELISA for rapid and sensitive clinical detection of ASFV antibodies.

African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that causes high mortality to domestic porcine and wild boars and brings huge economic losses to world swine industry. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, and cost-effective detection and strict control and elimination strategies. Traditional serological testing methods are generally associated with high testing costs, complex operations, and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity, although the system is dependent on the immunization of Bactrian camels to obtain the specific VHH library of the target protein. The application of Nbs in the detection of ASFV antibodies has not yet been reported yet. Using a phage display technology, one Nb against the ASFV p54 protein that exhibited high specificity and affinity, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. There was no cross-reaction with healthy pig serum, porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), and classical swine fever virus (CSFV) positive sera. The optimal cut-off value for the cELISA by ROC analysis was 52.5%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The results showed that the relative specificity of the cELISA was 98.97%, and the relative sensitivity of the cELISA was 93.3%, with the percent agreement between the two ELISA methods being 98.56%. In conclusion, a specific, sensitive, and repeatable cELISA was successfully developed based on the Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum. KEY POINTS: • We successfully screened a specific, high affinity nanobody against ASFV p54 protein. • We establish a method for continuous and stable expression of Nb-HRP fusion protein using a lentiviral packaging system. • We establish a nanobody cELISA detection method that can monitor an ASF infection.

Molecular Epidemiology and Pathogenic Characterization of Novel Chicken Infectious Anemia Viruses in Henan Province of China.

Chicken infectious anemia (CIA) is an immunosuppressive disease caused by the chicken infectious anemia virus (CIAV) resulting in heavy economic losses once an outbreak is established. This study conducted a systematic analysis of the epidemiology and pathology of CIA in Henan province, China. A total of 437 clinical tissue samples and 120 poultry disease-related live attenuated vaccines were collected during 2017-2020; of which 45 were positive for CIAV nucleic acid, with a positive rate of 8.08%. Our results showed that genome sequence similarity among a total of 12 CIAV isolates was high, and ranged from 97.1 to 99.3%, and their similarity to the vaccine strains Cux-1 and Del-Ros ranged from 97.8 to 98.6%. However, There were mutations in the locus of the major capsid proteins VP1, VP2, and VP3 among all isolates. The subsequent sequence analysis indicated that the isolates of HN-4 and HN-8 showed genetic recombination and follow up animal experiments revealed that HN-4 might be a pathogenic strain. Our results reveal that both field infection and non-CIAV vaccines contamination promote the epidemiology of CIAV in China and some dominant epidemic viruses have undergone recombination and evolution. This study provides important information to help with the prevention and control of CIAV in the poultry industry.

Deletion of miR-M8 and miR-M13 eliminates the bursa atrophy induced by Marek's disease virus infection.

Marek's disease (MD) is a neoplastic disease of chickens caused by an avian alphaherpesvirus, Marek's disease virus (MDV, also known as Gallid alphaherpesvirus 2 [GaHV2]). A total of 14 microRNA (miRNA) precursors and 26 mature miRNAs have been identified in MDV genome, which were grouped in three distinct clusters. In recent years, our studies revealed the role of MDV encoded cluster 3 miRNAs (or miR-M8-M10) and the specific function of its three members, miR-M6, miR-M7 and miR-M10, in regulating MDV replication and pathogenesis. In this study, we characterized the unique function of the other two members, miR-M8 and miR-M13, in cluster 3 miRNAs. Our results show that miR-M8 and miR-M13 are not important for MDV plaque formation and genome replication in vitro. Animal experiment results show that deletion of miR-M8-5p and miR-M13-5p eliminates the bursa atrophy, but not thymus atrophy, of MDV inoculated chickens. In addition, we found that the survival curve and MD incidences were not affected by disruption of miR-M8 and miR-M13. Taken together, this study uncovers the unique role of miR-M8 and miR-M13 in MDV replication and pathogenesis, which filled the gap in the research of MDV encoded miRNAs.

Identification and Characterization of a Novel Epitope of ASFV-Encoded dUTPase by Monoclonal Antibodies.

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) of African swine fever virus (ASFV) is an essential enzyme required for efficient virus replication. Previous crystallography data have indicated that dUTPase (E165R) may serve as a therapeutic target for inhibiting ASFV replication; however, the specificity of the targeting site(s) in ASFV dUTPase remains unclear. In this study, 19 mouse monoclonal antibodies (mAbs) were produced, in which four mAbs showed inhibitory reactivity against E165R recombinant protein. Epitope mapping studies indicated that E165R has three major antigenic regions: 100-120 aa, 120-140 aa, and 140-165 aa. Three mAbs inhibited the dUTPase activity of E165R by binding to the highly conserved 149-RGEGRFGSTG-158 amino acid sequence. Interestingly, 8F6 mAb specifically recognized ASFV dUTPase but not Sus scrofa dUTPase, which may be due to structural differences in the amino acids of F151, R153, and F154 in the motif V region. In summary, we developed anti-E165R-specific mAbs, and identified an important antibody-binding antigenic epitope in the motif V of ASFV dUTPase. Our study provides a comprehensive analysis of mAbs that target the antigenic epitope of ASFV dUTPase, which may contribute to the development of novel antibody-based ASFV therapeutics.

Marek's disease virus encoded miR-M6 and miR-M10 are dispensable for virus replication and pathogenesis in chickens.

MicroRNAs (miRNAs) are a class of approximately 22 nucleotides long non-coding RNAs, and virus-encoded miRNAs play an important role in pathogenesis. Marek's disease virus (MDV) is an oncogenic avian alphaherpesvirus that causes immunosuppression and tumors in its natural host, chicken. In the MDV genome, 14 miRNA precursors and 26 mature miRNAs were identified, thus MDV has been used as a model to study the function of viral miRNAs in vivo. Recently, a cluster of miRNAs encoded by MDV, Cluster 3 miRNAs (miR-M8-M10), has been shown to restrict early cytolytic replication and pathogenesis of MDV. In this study, we further analyzed the role of miR-M6 and miR-M10, members of cluster miR-M8-M10, in MDV replication and pathogenicity. We found that, compared to parental MDV, deletion of miR-M6-5p significantly enhanced the replication of MDV in cell culture, but not in chickens. The replication of miR-M6-5p deletion MDV was restored once the deleted sequences were re-inserted. Our results also showed that deletion of miR-M10-5p did not affect the replication of MDV in vitro and in vivo. In addition, our animal study results showed that deletion of miR-M6-5p or miR-M10-5p did not alter the pathogenesis of MDV. In conclusion, our study shows that both miR-M6 and miR-M10 are dispensable for MDV replication and pathogenesis in chickens, while also suggests a repressive role of miR-M6 in MDV replication in cell culture.

Virus-encoded microRNA-M7 restricts early cytolytic replication and pathogenesis of Marek's disease virus.

MicroRNAs (miRNAs) are a class of ∼22 nucleotides non-coding RNAs that are encoded by a wide range of hosts. Viruses, especially herpesviruses, encode a variety of miRNAs that involved in disease progression. Recently, a cluster of virus-encoded miRNAs, miR-M8-M10, have been shown to restrict early cytolytic replication and pathogenesis of Marek's disease virus (MDV), an oncogenic avian alphaherpesvirus that causes lymphoproliferative disease in chickens. In this study, we specifically dissected the role of miR-M7, a member of cluster miR-M8-M10, in regulating MDV replication and pathogenesis. We found that deletion of miR-M7-5p did not affect the virus plaque size and growth in cell culture. However, compared to parental virus, infection of miR-M7-5p deletion virus significantly increased MDV genome copy number at 5 days post infection, suggesting that miR-M7 plays a role to restrict MDV replication during early cytolytic phase. In addition, our results showed that infection of miR-M7-5p deletion virus significantly enhanced the mortality of chickens, even it induced lymphoid organ atrophy similar to parental and revertant viruses. Taken together, our study revealed that the miR-M7 acts as a repressive factor of MDV replication and pathogenesis.

Comprehensive profiling analysis of the N6-methyladenosine-modified circular RNA transcriptome in cultured cells infected with Marek's disease virus.

Marek's disease virus (MDV) induces severe immunosuppression and lymphomagenesis in the chicken, its natural host, and results in a condition that investigated the pathogenesis of MDV and have begun to focus on the expression profiling of circular RNAs (circRNAs). However, little is known about how the expression of circRNAs is referred to as Marek's disease. Previous reports have is regulated during MDV replication. Here, we carried out a comprehensive profiling analysis of N6-methyladenosine (m6A) modification on the circRNA transcriptome in infected and uninfected chicken embryonic fibroblast (CEF) cells. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) revealed that m6A modification was highly conserved in circRNAs. Comparing to the uninfected group, the number of peaks and conserved motifs were not significantly different in cells that were infected with MDV, although reduced abundance of circRNA m6A modifications. However, gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses revealed that the insulin signaling pathway was associated with the regulation of m6A modified circRNAs in MDV infection. This is the first report to describe alterations in the transcriptome-wide profiling of m6A modified circRNAs in MDV-infected CEF cells.

[A rapid and accurate method for herpesviral gnome editing].

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.

Transcriptome-wide N6-methyladenosine modification profiling of long non-coding RNAs during replication of Marek's disease virus in vitro.

BACKGROUND: The newly discovered reversible N6-methyladenosine (m6A) modification plays an important regulatory role in gene expression. Long non-coding RNAs (lncRNAs) participate in Marek's disease virus (MDV) replication but how m6A modifications in lncRNAs are affected during MDV infection is currently unknown. Herein, we profiled the transcriptome-wide m6A modification in lncRNAs in MDV-infected chicken embryo fibroblast (CEF) cells. RESULTS: Methylated RNA immunoprecipitation sequencing results revealed that the lncRNA m6A modification is highly conserved with MDV infection increasing the expression of lncRNA m6A modified sites compared to uninfected cell controls. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that lncRNA m6A modifications were highly associated with signaling pathways associated with MDV infection. CONCLUSIONS: In this study, the alterations seen in transcriptome-wide m6A occurring in lncRNAs following MDV-infection suggest this process plays important regulatory roles during MDV replication. We report for the first time profiling of the alterations in transcriptome-wide m6A modification in lncRNAs of MDV-infected CEF cells.