Splenic proteome profiling in response to Marek's disease virus strain GX0101 infection.

Marek's disease virus (MDV) strain GX0101 was the first reported field strain of recombinant gallid herpesvirus type 2 (GaHV-2). However, the splenic proteome of MDV-infected chickens remains unclear. In this study, a total of 28 1-day-old SPF chickens were intraperitoneally injected with chicken embryo fibroblast (CEF) containing 2000 PFU GX0101. Additionally, a control group, consisting of four one-day-old SPF chickens, received intraperitoneal equal doses of CEF. Blood and various tissue samples were collected at different intervals (7, 14, 21, 30, 45, 60, and 90 days post-infection; dpi) for histopathological, real-time PCR, and label-free quantitative analyses. The results showed that the serum expressions of MDV-related genes, meq and gB, peaked at 45 dpi. The heart, liver, and spleen were dissected at 30 and 45 dpi, and their hematoxylin-eosin staining indicated that virus infection compromised the normal organizational structure at 45 dpi. Particularly, the spleen structure was severely damaged, and the lymphocytes in the white medulla were significantly reduced. Furthermore, liquid chromatography-mass spectrometry (LC-MS) and label-free techniques were used to analyze the difference in splenic proteome profiles of the experimental and control groups at 30 and 45 dpi. Proteomic analysis identified 1660 and 1244 differentially expressed proteins (DEPs) at 30 and 40 dpi, respectively, compared with the uninfected spleen tissues. According to GO analysis, these DEPs were involved in processes such as organelle organization, cellular component biogenesis, cellular component assembly, anion binding, small molecule binding, metal ion binding, cation binding, cytosol, nuclear part, etc. Additionally, KEGG analysis indicated that the following pathways were linked to MDV-induced inflammation, apoptosis, and tumor: Wnt, Hippo, AMPK, cAMP, Notch, TGF-ß, PI3K-Akt, Rap1, Ras, Calcium, NF-κB, PPAR, cGMP-PKG, Apoptosis, VEGF, mTOR, FoxO, TNF, JAK-STAT, MAPK, Prion disease, T cell receptor, and B cell receptor. We finally screened 674 DEPs that were linked to MDV infection in spleen tissue. This study improves our understanding of the MDV response mechanism in the spleen.

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.

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.

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.

Study on the Dynamic Proliferation of JEV in BHK-21 Cells.

INTRODUCTION: Epidemic Japanese encephalitis is one of the most important zoonotic diseases that cause central nervous system damage. The vaccination has become the most effective and economical measure for its control. Hence, real-time monitoring of Japanese encephalitis virus (JEV) proliferation is crucial to optimize virus inoculation, culturing conditions, and virus harvest time. METHODS: The proliferation dynamics of JEV in BHK-21 cells was studied by combining the established quantitative PCR method with the conventional TCID50 assay in this study. RESULTS: The proliferation curve determined by the 2 methods has a definite parallel relationship, but the quantitative real-time PCR method (4 h) is faster and more sensitive than the TCID50 method (3-4 days). The determination results of TCID50 showed that the highest viral titer was 105.44 TCID50/0.1 mL and 104.86 TCID50/0.1 mL in cell suspension and culture supernate, respectively, while the virus RNA copies reached the peak at 1.0 × 107.5 copies/µL and 1.0 × 105.6 copies/µL in cell suspension and culture supernate, respectively. CONCLUSION: The comprehensive analysis showed that the best time for JEV proliferation in BHK-21 cell was 60 h post infection.

Purification of cell-derived Japanese encephalitis virus by dual-mode chromatography.

Purification of the enveloped virus poses a challenge as one must retain viral infectivity to preserve immunogenicity. The traditional process of virus purification is time-consuming, laborious and hard to scale up. Here, a rapid, simple and extensible laboratory program for the purification of Japanese encephalitis virus (JEV) was developed by using differential centrifugation, ultrafiltration, Sepharose 4 fast flow gel chromatography, and CaptoTM Core 700 chromatography. The entire process recovered 61.64% of the original virus, and the purified virus particles maintained good activity and immunogenicity. The purification process described has potential application in large-scale production of high-purity JEV.

Marek's Disease Virus-Encoded MicroRNA 155 Ortholog Critical for the Induction of Lymphomas Is Not Essential for the Proliferation of Transformed Cell Lines.

MicroRNAs (miRNAs) are small noncoding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV), respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and in lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, those studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative reverse transcription-PCR (qRT-PCR), and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is not essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumor cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gaining further insights into the biology of oncogenic herpesviruses.IMPORTANCE Marek's disease virus (MDV) is an alphaherpesvirus associated with Marek's disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype.

Phylogenetic analysis of porcine circovirus type 2 (PCV2) between 2015 and 2018 in Henan Province, China.

BACKGROUND: Porcine circovirus type 2 (PCV2) is the pathogen of porcine circovirus associated diseases (PCVAD) and one of the main pathogens in the global pig industry, which has brought huge economic losses to the pig industry. In recent years, there has been limited research on the prevalence of PCV2 in Henan Province. This study investigated the genotype and evolution of PCV2 in this area. RESULTS: We collected 117 clinical samples from different regions of Henan Province from 2015 to 2018. Here, we found that the PCV2 infection rate of PCV2 was 62.4%. Thirty-seven positive clinical samples were selected to amplify the complete genome of PCV2 and were sequenced. Based on the phylogenetic analysis of PCV2 ORF2 and complete genome, it was found that the 37 newly detected strains belonged to PCV2a (3 of 37), PCV2b (21 of 37) and PCV2d (13 of 37), indicating the predominant prevalence of PCV2b and PCV2d strains. In addition, we compared the amino acid sequences and found several amino acid mutation sites among different genotypes. Furthermore, the results of selective pressure analysis showed that there were 5 positive selection sites. CONCLUSIONS: This study indicated the genetic diversity, molecular epidemiology and evolution of PCV2 genotypes in Henan Province during 2015-2018.

Surface IgM λ light chain is involved in the binding and infection of infectious bursal disease virus (IBDV) to DT40 cells.

Infectious bursal disease virus (IBDV) is an important immunosuppressive virus in chickens. Surface immunoglobulin M (sIgM)-bearing B lymphocytes act as the major targets of IBDV in the bursa of Fabricius, and sIgM may function as one of the membrane binding sites responsible for IBDV infection. Recently, using the virus overlay protein binding assay, the chicken λ light chain of sIgM was identified to specifically interact with IBDV in a virulence-independent manner in vitro. To further investigate sIgM λ light chain-mediated IBDV binding and infection in pre-B cells, the cell line DT40, which is susceptible to both pathogenic and attenuated IBDV, was used. Based on the RNA interference strategy, the DT40 cell line whose λ light chain of sIgM was stably knocked down, herein termed DT40LKD, was generated by the genomic integration of a specific small hairpin RNA and a green fluorescence protein co-expression construct. Flow cytometry analysis indicated that the binding of IBDV to DT40LKD cells was significantly reduced due to the loss of sIgM λ light chain. In particular, reduced viral replication was observed in IBDV-incubated DT40LKD cells, and no viral release into cell culture medium was detected by the IBDV rapid diagnostic strips. In addition, the rescue of sIgM λ light chain expression restored viral binding and replication in DT40LKD cells. These results show that sIgM λ light chain appears to be beneficial for IBDV attachment and infection, suggesting that sIgM acts as a binding site involved in IBDV infection.

Hazy Weather-Induced Variation in Environmental Behavior of PCDD/Fs and PBDEs in Winter Atmosphere of A North China Megacity.

Haze is a serious pollution problem during the wintertime in North China. In this study, we investigated how the periodic cycle of winter haze events affect the environmental behaviors of two typical persistent organic pollutants, namely, polychlorinated dibenzo- p-dioxins and dibenzofurans (PCDD/Fs) and polybrominated diphenyl ethers (PBDEs), in the atmosphere of a typical megacity, Beijing. The average atmospheric concentrations of the total di- to octa-CDD/Fs (∑PCDD/Fs: 378.0 pg/m3) and the total mono- to nona-BDEs (∑9hPBDEs: 166.5 pg/m3) during haze episodes increased by 3.6-fold and 1.9-fold compared with those during the nonhaze periods, respectively; and their concentrations both linearly increased with PM2.5 levels and decreased as a power function of the atmospheric boundary layer height. The elevated concentrations could be clearly attributed to the vertically sinking motion of airflow in the midlower troposphere. When a haze event occurred, the partitioning rate of PCDD/Fs and PBDEs into particles was reduced; the largest fraction of the particle-bound ∑PCDD/Fs was shifted from ultrafine particles to accumulation mode particles; and a steady-state model (Li-Ma-Yang model) satisfactorily described the gas-particle partitioning of the PCDD/F and PBDE homologues. The inhalation exposure risk evaluation indicated that special attention should be paid to the increased cancer risk induced by the elevated inhalation intake of PCDD/Fs during haze episodes.

A SERS-based multiple immuno-nanoprobe for ultrasensitive detection of neomycin and quinolone antibiotics via a lateral flow assay.

The authors describe an ultrasensitive method for simultaneous detection of neomycin (NEO) and quinolones antibiotics (QNS). It is based on the use of (a) two immuno-nanoprobes (a probe for NEO and a probe for QNS), (b) surface-enhanced Raman scattering (SERS) detection, and (c), a portable lateral flow assay (LFA). The two probes consist of gold nanoparticles (AuNPs) conjugated to the Raman active molecule 4-aminothiophenol (PATP), and to monoclonal antibody against NEO (NEO mAb) or against NOR (NOR mAb). Quantitative detection of NEO and QNS was realized via SERS of the PATP-coated AuNPs captured in the test line of a LFA. Under optimized condition, the visual limits of LFA are 10 ng·mL-1 for NEO and 200 ng·mL-1 for NOR, and with LODs down to 0.37 pg·mL-1 and 0.55 pg·mL-1 by using SERS. The NEO test line is not interfered by the NEO analogues gentamycin, streptomycin and tobramycin, but the NOR test line suffers from different degrees of cross-reactivity (CR) to 12 common other QNS, the CRs ranging from 1.5% to 136%. The recoveries of NEO and NOR from spiked milk samples ranged between 86% and 121%, with relative standard deviations (RSD) from 3% to 6%. The method is highly sensitive, accurate and effective. It may be applied to simultaneous detection of NEO and 8 QNS, including NOR, enoxacin, ciprofloxacin, ofloxacin, fleroxacin, marbofloxacin, enrofloxacin, and pefloxacin. Graphical abstract Schematic of a lateral flow assay (LFA) based on an indirect competitive model. By using two test lines, the LFA can detect the neomycin and quinolones antibiotics simultaneously. Based on the surface-enhanced Raman scattering (SERS), the LFA shows high sensitivity to antibiotics with low limit of detection.

Comparison of two docking methods for peptide-protein interactions.

BACKGROUND: The importance of peptides in regulatory interactions has caused peptide-protein docking to attract the attention of many researchers. A variety of methods for molecular modeling of peptide-protein docking, such as local search and global search, are currently used. RESULTS: The interactions of 11 peptides and CSFV E2 protein were evaluated by the GalaxyPepDock and FlexX/ SYBYL programs, respectively. The assessment scores of all the peptides were correlated with their KD values. The final results showed that a moderate correlation coefficient was represented between KD values and CScores of predicted models by FlexX/ SYBYL. CONCLUSION: Our results demonstrate that considering the flexibility of the peptide is better than searching for more potential binding sites on the target protein surface while performing peptide-protein molecular docking. These data provide reasonable evidence for the molecular design of peptides and guidance for the functional assignment of target proteins. © 2018 Society of Chemical Industry.

Putative roles as oncogene or tumour suppressor of the Mid-clustered microRNAs in Gallid alphaherpesvirus 2 (GaHV2) induced Marek's disease lymphomagenesis.

In the last decade, numerous microRNAs (miRNAs) have been identified in diverse virus families, particularly in herpesviruses. Gallid alphaherpesvirus 2 (GaHV2) is a representative oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts, namely Marek's disease (MD). In the GaHV2 genome there are 26 mature miRNAs derived from 14 precursors assembled into three clusters, namely the Meq-cluster, Mid-cluster and LAT-cluster. Several GaHV2 miRNAs, especially those in the Meq-cluster (e.g. miR-M4-5p), have been demonstrated to be critical in MD pathogenesis and/or tumorigenesis. Interestingly the downstream Mid-cluster is regulated and transcribed by the same promoter as the Meq-cluster in the latent phase of the infection, but the role of these Mid-clustered miRNAs in GaHV2 biology remains unclear. We have generated the deletion mutants of the Mid-cluster and of its associated individual miRNAs in GX0101 virus, a very virulent GaHV2 strain, and demonstrated that the Mid-clustered miRNAs are not essential for virus replication. Using GaHV2-infected chickens as an animal model, we found that, compared with parental GX0101 virus, the individual deletion of miR-M31 decreased the mortality and gross tumour incidence of infected chickens while the deletion individually of miR-M1 or miR-M11 unexpectedly increased viral pathogenicity or oncogenicity, similarly to the deletion of the entire Mid-cluster region. More importantly, our data further confirm that miR-M11-5p, the miR-M11-derived mature miRNA, targets the viral oncogene meq and suppresses its expression in GaHV2 infection. We report here that members of the Mid-clustered miRNAs, miR-M31-3p and miR-M11-5p, potentially act either as oncogene or tumour suppressor in MD lymphomagenesis.

Colloidal gold-McAb probe-based rapid immunoassay strip for simultaneous detection of fumonisins in maize.

BACKGROUND: Fumonisins are a kind of toxic and carcinogenic mycotoxin. A rapid immunochromatographic test strip has been developed for simultaneous detection of fumonisin B1 , B2 and B3 (FB1 , FB2 and FB3 ) in maize based on colloidal gold-labelled monoclonal antibody (McAb) against FB1 probe. RESULTS: The anti-FB1 McAb (2E11-H3) was produced through immunisation and cell fusion, and identified as high affinity, specificity and sensitivity. The cross-reaction ratios with fumonisin B2 and B3 were accordingly 385% and 72.4%, while none with other analogues. The colloid gold-labelled anti-FB1 McAb probe was successfully prepared and used for establishing the immunochromatographic strip. The test strip showed high sensitivity and specificity, the IC50 for FB1 was 58.08 ng mL-1 , LOD was 11.24 ng mL-1 , calculated from standard curve. Moreover, the test strip exhibited high cross-reactivity with FB2 and FB3 , and could be applied to the simultaneous detection of FBs (FB1 :FB2 :FB3 = 12:4:1) in maize sample with high accuracy and precision. The average recoveries of FBs in maize ranged from 90.42% to 95.29%, and CVs were 1.25-3.77%. The results of the test strip for FBs samples showed good correlation with high-performance liquid chromatography analysis. CONCLUSION: The immunochromatographic test strip could be employed in the rapid simultaneous detection of FB1 , FB2 and FB3 in maize samples on-site. © 2016 Society of Chemical Industry.

The significance of the individual Meq-clustered miRNAs of Marek's disease virus in oncogenesis.

Marek's disease virus (MDV) is an important oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts. The Meq-clustered miRNAs encoded by MDV have been suggested to play potentially critical roles in the induction of lymphomas. Using the technique of bacterial artificial chromosome mutagenesis, we have presently constructed a series of specific miRNA-deleted mutants and demonstrate that these miRNAs are not essential for replication of MDV and have no effects on the early cytolytic or latent phases of the developing disease. However, compared to the parental GX0101, mortality of birds infected with the mutants GXΔmiR-M2, GXΔmiR-M3, GXΔmiR-M5, GXΔmiR-M9 and GXΔmiR-M12 was reduced from 100 % to 18 %, 30 %, 48 %, 24 % and 14 %, coupled with gross tumour incidence reduction from 28 % to 8 %, 4 %, 12 %, 8 % and 0 %, respectively. Our data confirm that except for mdv1-miR-M4, the other Meq-clustered miRNAs also play critical roles in MDV oncogenesis. Further work will be needed to elucidate the miRNA-mediated regulatory mechanisms that trigger the development of MD lymphomas.

In vivo expression patterns of microRNAs of Gallid herpesvirus 2 (GaHV-2) during the virus life cycle and development of Marek's disease lymphomas.

In the past decade, a large number of microRNAs (miRNAs) have been identified in the viral genome of Gallid herpesvirus 2 (GaHV-2), which is historically known as Marek's disease virus type 1. The biological role of most GaHV-2 miRNAs remains unclear. In the present study, we have performed an overall gene expression profile of GaHV-2 miRNAs during the virus life cycle at each phase of the developing disease, a highly contagious, lymphoproliferative disorder, and neoplastic immunosuppressive disease of poultry known as the Marek's disease. According to their distinct in vivo expression patterns, the GaHV-2 miRNAs can be divided into three groups: 12 miRNAs in group I, including miR-M4-5p, displayed a typical expression pattern potentially correlated to the latent, late cytolytic, and/or the proliferative phases in the cycle of GaHV-2 pathogenesis; group II consisting of another 12 miRNAs with expression correlated to the early cytolytic and/or latent phases in GaHV-2's life cycle; while the other two miRNAs in group III showed no identical expression features. Our findings may provide meaningful clues in the search for further potential functions of viral miRNAs in GaHV-2 biology.

Viral miRNA delivered by exosomes from Marek's disease virus-transformed lymphoma cell line exerts regulatory function in internalized primary chicken embryo fibroblast cells.

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.

Immune escape of avian oncogenic Marek's disease herpesvirus and antagonistic host immune responses.

Marek's disease virus (MDV) is a highly pathogenic and oncogenic alpha herpesvirus that causes Marek's disease (MD), which is one of the most important immunosuppressive and rapid-onset neoplastic diseases in poultry. The onset of MD lymphomas and other clinical diseases can be efficiently prevented by vaccination; these vaccines are heralded as the first demonstration of a successful vaccination strategy against a cancer. However, the persistent evolution of epidemic MDV strains towards greater virulence has recently resulted in frequent outbreaks of MD in vaccinated chicken flocks worldwide. Herein, we provide an overall review focusing on the discovery and identification of the strategies by which MDV evades host immunity and attacks the immune system. We have also highlighted the decrease in the immune efficacy of current MD vaccines. The prospects, strategies and new techniques for the development of efficient MD vaccines, together with the possibilities of antiviral therapy in MD, are also discussed.

Molecular characterization of Japanese encephalitis viruses circulating in pigs and mosquitoes on pig farms in the Chinese province of Henan.

Since the first Chinese case report of Japanese encephalitis, Japanese encephalitis virus (JEV) has circulated in China for at least 60 years. Even though pigs play a critical role in the JEV transmission cycle information on the prevalence of JEV in pigs has not been investigated in China. As the central Chinese province of Henan has the largest human population in China, a history of serious JEV and is the largest pig producing province it was chosen for this study. We have found that currently natural infection with JEV in pigs and mosquitoes is prevalent and both genotypes 1 and 3 co-circulate in pigs and mosquitoes in central China. Phylogenetic analysis showed that all of the newly obtained pig-derived JEV isolates are more closely related to isolates from the 1950s to 1960s than to those recently isolated from humans and mosquitoes. Further analyses based on all the previous reported Chinese isolates indicates that presently genotype 3 JEV is the predominant genotype in pigs but genotype 1 JEV is emerging and spreading rapidly in recent years. Our study provides information for understanding the current epidemiology of JEV in China and suggests possible measures applicable to the further control of JEV.

Molecular characteristics and evolutionary analysis of field Marek's disease virus prevalent in vaccinated chicken flocks in recent years in China.

Marek's disease is a highly contagious, oncogenic, and immunosuppressive avian viral disease. Surveillance of newly registered Marek's disease virus (MDV) isolates is meaningful for revealing the potential factors involved in increased virulence. Presently, we have focused on the molecular characteristics of all available MDVs from China, including 17 new Henan isolates. Based on Meq, gE, and gI genes, we found that most Chinese isolates contain conserved amino acid point mutations in Meq, such as E(77), A(115), A(139), R(176), and A(217), compared to USA virulent MDVs. However, the 59-aa or 60-aa insertions are only found in a few mild MDVs rather than virulent MDVs in China. Further phylogenetic analysis has demonstrated that a different genotype of MDV has been prevalent in China, and for virulent MDVs, their recent evolution has possibly been geographically restricted. Our study has provided more detailed information regarding the field MDVs circulating in China.