Shuai Su

Deletion of the meq gene significantly decreases immunosuppression in chickens caused by pathogenic marek's disease virus

BackgroundMareks disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in immunosuppression, which is considered to be an integral aspect of the pathogenesis of Mareks disease (MD). A recent study showed that deletion of the Meq gene resulted in loss of transformation of T-cells in chickens and a Meq-null virus, rMd5ΔMeq, could provide protection superior to CVI988/Rispens.ResultsIn the present study, to investigate whether the Meq-null virus could be a safe vaccine candidate, we constructed a Meq deletion strain, GX0101ΔMeq, by deleting both copies of the Meq gene from a pathogenic MDV, GX0101 strain, which was isolated in China. Pathogenesis experiments showed that the GX0101ΔMeq virus was fully attenuated in specific pathogen-free chickens because none of the infected chickens developed Mareks disease-associated lymphomas. The study also evaluated the effects of GX0101ΔMeq on the immune system in chickens after infection with GX0101ΔMeq virus. Immune system variables, including relative lymphoid organ weight, blood lymphocytes and antibody production following vaccination against AIV and NDV were used to assess the immune status of chickens. Experimental infection with GX0101ΔMeq showed that deletion of the Meq gene significantly decreased immunosuppression in chickens caused by pathogenic MDV.ConclusionThese findings suggested that the Meq gene played an important role not only in tumor formation but also in inducing immunosuppressive effects in MDV-infected chickens.

Complete Genome Sequence of a Recombinant Marek's Disease Virus Field Strain with One Reticuloendotheliosis Virus Long Terminal Repeat Insert

ABSTRACT Mareks disease virus (MDV) Chinese strain GX0101, isolated in 2001 from a vaccinated flock of layer chickens with severe tumors, was the first reported recombinant MDV field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. GX0101 belongs to very virulent MDV (vvMDV) but has higher horizontal transmission ability than the vvMDV strain Md5. The complete genome sequence of GX0101 is 178,101 nucleotides (nt) and contains only one REV-LTR insert at a site 267 nt upstream of the sorf2 gene. Moreover, GX0101 has 5 repeats of a 217-nt fragment in its terminal repeat short (TRS) region and 3 repeats in internal repeat short (IRS) region, compared to the other 10 strains with only 1 or 2 repeats in both TRS and IRS.

Genomic sequence analysis and biological characteristics of a rescued clone of avian leukosis virus strain JS11C1, isolated from indigenous chickens.

The strain JS11C1, a member of a putative new subgroup of avian leukosis virus (ALV) that is different from all six known subgroups from chickens based on Gp85 amino acid sequence comparison, was isolated from Chinese native chicken breeds in 2012. In order to further study the genome structure, biological characteristics, and the evolutionary relationship of the virus with others of known subgroups from infected chickens, we determined the complete genome sequence, constructed an infectious clone of ALV strain JS11C1, and performed comparative analysis using the whole genome sequence or elements with that of other ALVs available in GenBank. The results showed that the full-length sequence of the JS11C1 DNA provirus genome was 7707 bp, which is consistent with a genetic organization typical of a replication-competent type C retrovirus lacking viral oncogenes. The rescued infectious clone of JS11C1 showed similar growth rate and biological characteristics to its original virus. All the comparison analyses based on whole genomes support the opinion that the new isolates are relatively distantly related to any known subgroups of ALVs and might be classified as a new subgroup.

A recombinant field strain of Marek's disease (MD) virus with reticuloendotheliosis virus long terminal repeat insert lacking the meq gene as a vaccine against MD

Mareks disease virus (MDV) GX0101, which is a field strain with a naturally occurring insertion of the reticuloendotheliosis virus (REV) long terminal repeat (LTR) fragment, shows distinct biological activities. Deletion of the meq gene in GX0101 contributes to its complete loss of pathogenicity and oncogenicity in SPF chickens, but this virus has a kanamycin resistance gene (kan(r)) residual at the site of meq gene. In the present study, the kan(r) was knocked out and a meq-null virus with a good replicative ability termed SC9-1 was selected. In vivo studies showed that SC9-1 had no pathogenicity or tumorigenicity to chickens. There were no obvious impacts on chicken weight, immune organ index or antibody levels induced by avian influenza virus (AIV)/newcastle disease virus (NDV) inactivated vaccines compared with the control group. The SC9-1 virus provided superior protection than CVI988/Rispens vaccine in both SPF chickens and Hy-line brown chickens when challenged with a very virulent MDV (rMd5 strain). There was no obvious change in SC9-1 protection against MDV rMd5 in SPF chickens after 20 passages in chicken embryonic fibroblast cells (CEFs). In conclusion, SC9-1 is a safe and effective vaccine candidate for the prevention of Mareks disease.

Isolation and pathogenic analysis of virulent Marek's disease virus field strain in China.

Mareks disease (MD) has become increasingly common in China, resulting in considerable economic loss. The etiological agent is unclear. In this study, we isolated a field MD virus (MDV) strain, designated SX1301, from CVI988/Rispens-vaccinated chickens with tumors. Co-infection of avian leukosis virus, reticuloendotheliosis virus, and chicken infectious anemia virus was excluded by polymerase chain reaction, enzyme-linked immunosorbant assay, DNA blotting hybridization, and indirect immunofluorescence assay. As with most strains isolated in China, SX1301 had the same amino acid mutation of meq protein at positions 77(E), 80(Y), and 115(A) Animal experimental results showed development of lethal MD in 57% and MD tumor in 23% of the specific pathogen-free chickens inoculated with SX1301, with tumors mainly distributed in spleen, liver, and kidney. CVI988/Rispens protected 83% of chickens upon challenge with SX1301, with a mortality rate and tumor incidence of 10% and 7%, respectively. These results implicated SX1301 as a virulent MDV strain, with commercial MDV vaccine CVI988/Rispens unable to confer adequate protection against SX1301. There have been no reports of very virulent (vv) plus MDV in China, but frequently occurring virulent MDV may account for the repeated outbreaks of MD. Vaccines with greater efficacy are needed to protect against MDV.

Deletion of 1.8-kb mRNA of Marek's disease virus decreases its replication ability but not oncogenicity

BackgroundThe 1.8-kb mRNA was reported as one of the oncogenesis-related genes of Mareks disease virus (MDV). In this study, the bacterial artificial chromosome (BAC) clone of a MDV field strain GX0101 was used as the platform to generate mutant MDV to examine the functional roles of 1.8-kb mRNA.ResultsBased on the BAC clone of GX0101, the 1.8-kb mRNA deletion mutant GX0101Δ(A+C) was constructed. The present experiments indicated that GX0101Δ(A+C) retained a low level of oncogenicity, and it showed a decreased replication capacity in vitro and in vivo when compared with its parent virus, GX0101. Further studies in vitro demonstrated that deletion of 1.8-kb mRNA significantly decreased the transcriptional activity of the bi-directional promoter between 1.8-kb mRNA and pp38 genes of MDV.ConclusionThese results suggested that the 1.8-kb mRNA did not directly influence the oncogenesis but related to the replication ability of MDV.

Synergy of subgroup J avian leukosis virus and Eimeria tenella to increase pathogenesis in specific-pathogen-free chickens

To investigate the effects of co-infections of subgroup J avian leukosis virus (ALV-J) and Eimeria tenella on the pathogenesis in specific-pathogen-free (SPF) white leghorn chickens, groups of chickens were infected with ALV-J strain NX0101 at one day of age or with E. tenella at 14 days of age or both. The control group was left uninfected and was mock-inoculated with phosphate buffer saline (PBS). Mortality rates, body weights, cecal lesions, and viremia of infected chickens in each group were evaluated. Immune status was evaluated by measuring several parameters: immune organ weight/body weight index, specific humoral responses to inactivated NDV vaccine and to inoculated E. tenella, proportions of blood CD3+CD4+ and CD3+CD8α+ lymphocytes and transcriptional levels of cytokines in blood and cecal tonsils. The results show that co-infections of ALV-J and E. tenella induced a higher mortality rate and a lower body weight in SPF chickens compared to single-pathogen infection. In co-infected chickens, ALV-J accelerated the disease symptoms induced by E. tenella, and the E. tenella extended the ALV-J viremia. Thymus atrophy, decrease in the humoral response levels to pathogens and the NDV vaccine, modifications in the blood lymphocyte sub-populations and transcriptional cytokine disorders were found in co-infected chickens compared to chickens infected with one pathogen alone and to controls. We underline a synergy between ALV-J and E. tenella that results in increasing pathogenesis in SPF chickens.

Transcriptional and Bioinformatic Analysis Provide a Relationship between Host Response Changes to Marek's Disease Viruses Infection and an Integrated Long Terminal Repeat.

GX0101, Mareks disease virus (MDV) strain with a long terminal repeat (LTR) insert of reticuloendotheliosis virus (REV), was isolated from CVI988/Rispens vaccinated birds showing tumors. We have constructed a LTR deleted strain GX0101ΔLTR in our previous study. To compare the host responses to GX0101 and GX0101ΔLTR, chicken embryo fibroblasts (CEF) cells were infected with two MDV strains and a gene-chip containing chicken genome was employed to examine gene transcription changes in host cells in the present study. Of the 42,368 chicken transcripts on the chip, there were 2199 genes that differentially expressed in CEF infected with GX0101 compared to GX0101ΔLTR significantly. Differentially expressed genes were distributed to 25 possible gene networks according to their intermolecular connections and were annotated to 56 pathways. The insertion of REV LTR showed the greatest influence on cancer formation and metastasis, followed with immune changes, atherosclerosis, and nervous system disorders in MDV-infected CEF cells. Based on these bio functions, GX0101 infection was predicated with a greater growth and survival inhibition but lower oncogenicity in chickens than GX0101ΔLTR, at least in the acute phase of infection. In summary, the insertion of REV LTR altered the expression of host genes in response to MDV infection, possibly resulting in novel phenotypic properties in chickens. Our study has provided the evidence of retroviral insertional changes of host responses to herpesvirus infection for the first time, which will promote to elucidation of the possible relationship between the LTR insertion and the observed phenotypes.

Depression of Vaccinal Immunity to Marek’s Disease by Infection with Chicken Infectious Anemia Virus

Marek’s disease (MD) has been occurring with increasing frequency in chickens in recent years. To our knowledge, however, there has been no report of the very virulent plus (vv+) MD virus (MDV) field isolate in China. Studies have shown that dual infection with immunosuppressive viruses such as chicken infectious anemia virus (CIAV) occurs frequently in chickens developing MD. In this study, we performed a designed set of in vivo experiments, which comprised five different groups of chickens, including the group of CVI988/Rispens-vaccinated chickens, the groups of CVI988/Rispens-vaccinated chickens infected with MDV or CIAV or both viruses (MDV and CIAV), and the group of MDV-challenged chickens. The effects of CIAV dual infection on the immunization of commercial MDV vaccine CVI988/Rispens were evaluated. The results show that infection of the SD15 strain of CIAV significantly reduced the weight and antibody titers to avian influenza virus (AIV)/Newcastle disease virus (NDV) inactivated vaccines of chickens immunized with the CVI988/Rispens, and resulted in the atrophy of thymus/bursa and the enlargement of spleen. The CVI988/Rispens vaccination conferred good immune protection for chickens challenged with 2000 PFU of the GX0101 strain of MDV. However, dual infection with SD15 significantly reduced the body weight, antibody titers induced by AIV/NDV inactivated vaccines and protective index of CVI988/Rispens, and resulted in the aggravation of the immunosuppression, mortality, and viremia of GX0101 in CVI988/Rispens-immunized/GX0101-challenged chickens. Overall, CIAV infection significantly reduced the protective effects of the CVI988/Rispens vaccine against MDV, implying that concurrent infection with CIAV may be a major contributor in the frequent attacks of MD in China in recent years.

Deletion of the BAC sequences from recombinant meq-null Marek's disease (MD) virus increases immunosuppression while maintaining protective efficacy against MD

Mareks disease virus (MDV) GX0101 is a field strain of MDV with a naturally occurring insertion of the reticuloendotheliosis virus (REV) long terminal repeat (LTR) fragment. Both copies of the meq gene were knocked out in the GX0101 bacterial artificial chromosome (BAC) clone to construct the recombinant virus SC9-1, resulting in a complete lack of pathogenicity and providing better protection against MD than CVI988/Rispens. In the present study, the BAC sequences in SC9-1 were removed using a cre-loxP system, and the virus termed SC9-2. SC9-2 showed a significant increase in replication in vitro and in vivo. There was a significant decrease in chicken weight, immune organ index, and antibody levels compared with those of SC9-1-inoculated chickens. The immune protection index of SC9-2 was similar to that of SC9-1, and the difference was not significant. The results of our studies demonstrate that the SC9-2 virus provides protection in specific pathogen free (SPF) chickens when challenged with a very virulent MDV rMd5, but it induces immunosuppressive effects in SPF chickens.