Dynamic Co-evolution and Interaction of Avian Leukosis Virus Genetic Variants and Host Immune Responses.

Subgroup J avian leukosis virus (ALV-J), a typical retrovirus, is characterized of existence of a cloud of diverse variants and considerable genetic diversity. Previous studies describing the evolutionary dynamics of ALV-J genetic variants mainly focused on the early infection period or few randomly selected clones. Here, we inoculated 30 specific-pathogen-free chickens with the same founder ALV-J stock of known genetic background. Six (three antibody positive and three antibody negative) chickens were selected among 15 chickens with viremia. Viruses were serially isolated in 36 weeks and then sequenced using MiSeq high-throughput sequencing platform. This produced the largest ALV-J dataset to date, composed of more than three million clean reads. Our results showed that host humoral immunity could greatly enhance the genetic diversity of ALV-J genetic variants. In particular, selection pressures promoted a dynamic proportional changes in ALV-J genetic variants frequency. Cross-neutralization experiment showed that along with the change of the dominant variant, the antibody titers specific to infectious clones corresponding to the most dominant variants in weeks 12 and 28 have also changed significantly in sera collected in weeks 16 and 32. In contrast, no shift of dominant variant was observed in antibody-negative chickens. Moreover, we identified a novel hypervariable region in the gp85 gene. Our study reveals the interaction between ALV-J and the host, which could facilitate the development of vaccines and antiviral drugs.

Lamivudine Inhibits the Replication of ALV-J Associated Acutely Transforming Virus and its Helper Virus and Tumor Growth In vitro and In vivo.

To study the antiviral effects of lamivudine on avian leukosis virus subgroup J (ALV-J) and its inhibitory effect on the growth of fibrosarcomas caused by acute transforming avian leukosis virus, a series of experiments were performed in chicken embryo fibroblast cultures and 1-day-old chickens inoculated with an acutely transforming viral stock Fu-J (SDAU1005). This stock was prepared from an acutely fibrosarcoma of field cases in chicken farms and contained both the replication-defective virus Fu-J carrying v-fps oncogene and its helper virus ALV-J strain SDAU1005. The results from three different assays in cell cultures demonstrated the significant inhibitory effect of lamivudine on the replication of both SDAU1005 and Fu-J viruses. Furthermore, the effect was dose dependent in the concentration range of 1-4 µg/ml. In chicken experiments, lamivudine could decrease the viral loads of SDAU1005 and Fu-J in the plasma of inoculated chickens, delay the appearance of acute sarcomas, and decrease chicken mortality in the early stage. This model may be used to directly evaluate the inhibitory effects of lamivudine on such tumors and to understand the relationship between the replication-defective virus and its helper virus while also assessing tumor processes.

[Construction of Recombinant Marek's Disease Virus Expressing the NDV-F gene and its Replication in Chickens and in Vitro].

We used a meq-deleted attenuated MDV-I strain GX0101Δmeq as a vector to construct a recombinant virus expressing the exogenous gene NDV-F. The ORF of exogenous gene NDV-F was inserted into the eukaryotic expression vector pcDNA3.1(-). Then, the expression cassette of NDV-F which contains the CMV promoter was amplified. Simultaneously, we amplified the selected gene Kan+ expression cassette and inserted them into the PMD18-T vector. Tandem expression cassettes were amplified using primers containing the 50-bp homologous arm of MDV-US2. The PCR product was electroporated into EL250 host bacteria containing GX0101Δmeq. Then, the Kan+ expression cassette was deleted from the recombinant virus genome using 1% arabinose. The plasmid of the positive clone which the Kan+ expression cassette was deleted was extracted and transfected into CEFs to rescue the recombinant virus. The recombinant virus was injected into chickens to observe its growth and replication. The recombinant virus rMDV-F containing the exogenous gene NDV-F was rescued successfully. The recombinant virus could duplicate and express well in CEFs, and grow and replicate well in chickens. Using GX0101Δmeq as a vector, combined with a recombinant system of Red E/T and FLP/FRT, we constructed a recombinant virus that expressed the exogenous gene NDV-F. This study could lay the foundation for further study of recombinant viruses.

Transcriptional activity comparison of different sites in recombinant Marek's disease virus for the expression of the H9N2 avian influenza virus hemagglutinin gene.

Over the last two decades, much attention has been paid to MDV-vectored recombinant vaccines. Many factors have influenced their protective efficacy, and insertion site has been among the main influential factors for the expression of foreign genes in recombinant Marek's disease virus (rMDV). To compare the transcriptional activity of different sites of rMDV, an H9N2 avian influenza virus hemagglutinin gene (AIV-H9N2-HA) expression cassette that used the bi-directional promoter of serotype 1 MDV (MDV1) in the 1.8kb RNA transcript direction (p1.8kb) as a promoter was inserted into 4 different regions of MDV using the bacterial artificial chromosome (BAC) vector and FLP/FRT recombination technique. The insertion regions included 3 of its own sites (US2, US10 and one of Meq genes) in the MDV genome and a foreign site (gpt gene) in the BAC vector. Quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to analyze and compare the H9N2-HA expression levels of these different rMDVs both at the mRNA level and at the protein level. The results indicated that among the four tested insertion regions, the HA expression cassette in the US2 region demonstrated the highest activity, followed by that in the Meq region, which was almost equal to that of US10. Further, the expression cassette had the lowest activity in the foreign region gpt gene. The above data could be useful for choosing proper recombinant insertion regions in the construction of rMDV to express different foreign genes, and it is a prerequisite for developing effective MDV-vectored recombinant vaccines.

Construction of recombinant Marek's disease virus (MDV) lacking the meq oncogene and co-expressing AIV-H9N2 HA and NA genes under control of exogenous promoters.

To develop a recombinant Marek's disease virus (rMDV1) co-expressing the hemagglutinin gene (HA) and neuramidinase gene (NA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain and lacking the meq oncogene that shares homology with the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101 was used as parental virus, the HA and NA genes co-expression cassette under control of the CMV and SV40 early promoters was inserted at two meq sites of GX0101 to form a new meq knock-out mutant MDV (MZC12HA/NA) through homologous recombination. MZC12HA/NA was reconstituted by transfection of recombinant BAC-MDV DNA into the secondary chicken embryo fibroblast (CEF) cells. Highly purified MZC12HA/NA was obtained after four rounds of plaque purification and proliferation. In vitro growth properties of recombinant virus were also inspected and concluded that the MZC12HA/NA had the same growth kinetics in CEF cultures as its parental wild type virus GX0101. Southern blot indicated that co-expression cassette was successfully inserted at two copies sites of meq gene, so two meq genes were knocked-out completely. RT-qPCR showed transcription and expression levels of the HA and NA genes were both significantly higher than that of GX0101 own pp38 gene. Indirect fluorescence antibody (IFA) test, and Western blot analyses indicated that HA and NA genes were co-expressed simultaneously under control of the different promoters but meq genes were not. These results herald a new and effective recombinant meq-deleted MDV-based AIV-H9N2 vaccine may be useful in protecting chickens from very virulent MDV and H9N2 challenges.

[Comparison of immunoprotection between vaccination with meq-deleted Marek's disease virus and vaccine strain CVI988/Rispens].

OBJECTIVE: To evaluate and compare the immunoprotection between a meq-deleted Marek's disease virus (MDV) and CVI988/Rispens against MDV very virulent strain GX0101. METHODS: In total 120 one-day-old SPF chickens were divided into 4 groups (30 each) and kept in 5 isolators with positive pressure-filtered air. At 1 day of age, 2000 PFU of SC9-1 was inoculated subcutaneously into each bird in group 1; 2000 PFU of commercial vaccine CVI988/ Rispens was inoculated subcutaneously into each bird in group 2. No viral challenge was made in group 3 and 4 as controls. Five days later chickens in group 1, 2, 3 were challenged intra-abdominally with 2000 PFU of very virulent MDV strain GX0101. During 90 days after challenge, all dead birds were recorded and checked for necropsy. The tumor-suspected tissues were examined by histopathological biopsy. The antibody titers induced by AIV and NDV vaccination and propagation dynamics of MDV GX0101 were detected. At the same time, parallel tests were performed on Hy-Line Brown chickens containing MDV maternal antibody. RESULTS: SC9-1 stain provided 100% protective efficiency against very virulent GX0101 challenge in SPF and Hy-Line Brown chickens. CVI988/Rispens provided 86. 7% protective efficiency against very virulent GX0101 challenge in SPF chickens and 93% in Hy-Line Brown chickens. Challenge with GX0101 caused 53.3% mortality and 16.7% of birds with gross tumors in SPF chickens while there was 36.7% mortality and 16.7% of birds with gross tumors in Hy-Line Brown chickens, and there was no tumor lesion in histopathological biopsy in control group. The results of qPCR demonstrated that the copies of GX0101 viral genomes in SC9-1 vaccinated chickens was lower than CVI988/Rispens vaccinated chickens in lymphocyte and feather follicle DNA. The results of hemagglutination inhibition test demonstrated that antibody titers of AIV and NDV was higher in SC9-1 vaccinated chickens than that in CVI988/Rispens vaccinated chickens. CONCLUSION: SC9-1 stain's immunoprotection against MDV is more effective than CVI988/Rispens strain's both in SPF chickens and commercial Hy-Line Brown chickens containing maternal antibody.