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.

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.

Expression profiles of microRNAs encoded by the oncogenic Marek's disease virus reveal two distinct expression patterns in vivo during different phases of disease.

Marek's disease virus (MDV) is a long-recognized oncogenic herpesvirus, which induces lymphoma in its natural host that can be prevented by vaccination. MDV infection provides an excellent biological model for investigating the biology, genetics and immunology of viral oncogenesis. Recently discovered microRNAs (miRNAs) in the MDV genome have been suggested to have regulatory roles during MDV oncogenesis. We have examined the expression profiles of all 22 previously reported miRNAs encoded by MDV-1 in chickens artificially challenged with MDV-GX0101. We found that a subset of the miRNAs was differentially expressed during different phases of the developing disease. These miRNAs show early or late expression during disease progression, accompanied by obvious tissue-specific and differential expression patterns. This temporal and differential tissue distribution suggest that these miRNAs may perform different regulatory roles in switching from latency to lytic replication, immunosuppression, neoplastic transformation or other aspects of lymphoma formation. These reported in vivo expression profiles indicate the potentially functional MDV-1-encoded miRNAs that should be selected for further investigation of their functions in MDV oncogenesis.

Molecular epidemiological investigation of Marek's disease virus from Guangxi, China.

The predominant field strains of Marek's disease virus in Guangxi were clearly different from the vaccine strain CVI988/Rispens based on sequencing of the envelope glycoprotein I (gI), glycoprotein E (gE) and oncogenic meq genes. These differences may be partly responsible for the most recent outbreaks in Guangxi.

Molecular characterization of three new virulent Newcastle disease virus variants isolated in China.

Three cases of Newcastle disease virus (NDV) found in nature had the lentogenic motif (112)G-R-Q-G-R-L(117) in their fusion protein cleavage sites. However, both intracerebral pathogenicity and intravenous pathogenicity indexes showed that these NDV isolates were virulent. In comparison with the LaSota live virus vaccine, these viruses had significant genetic variations in the hemagglutinin-neuraminidase gene.

Effects of reticuloendotheliosis virus and Marek's disease virus infection and co-infection on IFN-gamma production in SPF chickens.

Two experiments were used to examine the potential role of IFN-gamma in chickens infected with reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). First, chickens were infected with REV and/or MDV at 5 days of age and examined from 3 to 50 days post-infection (dpi). In REV+MDV co-infection chickens, IFN-gamma ELISA demonstrated a 3-fold increase at 7 dpi compared to the controls, while REV alone caused a 5-fold increase, the IFN-gamma levels peaked, and then gradually decreased. IFN-gamma levels significantly decreased in MDV infection at 3 dpi and 15 dpi. Second, experiments were designed to determine the effects of different viruses and ConA on IFN-gamma production. For REV- or MDV-infected chickens, the IFN-gamma levels decreased slightly after adding ConA. This is the first report of IFN-gamma production in SPF chickens infected with REV and MDV measured by directly quantitative method.

[Pathogenicity and genomic sequence comparison of a chicken infectious anemia virus field isolate].

A field strain C14 of chicken infectious anemia virus (CAV) was isolated from a 14 day-old broiler flock with growth runting syndromes. Antibody reactions to inactivated vaccines to avian influenza viruses (AIV) were suppressed in SPF chickens inoculated with C14 strain CAV at 1 day-old. Also C14 strain CAV and reticuloendotheliosis Virus demonstrated a synergism in immunosuppression when chickens were infected with both virus. The viral genomic DNA was amplified by PCR in 3 overlapped fragments and PCR products were cloned into T-vector plasmid for sequencing. The sequencing results indicated that the total genome of C14 strain CAV was 2298bp, it contained 3 overlapped ORF and 1 non-coding regulation fragment. Its whole genome had 97.2% - 99.2% of homogeneity to other several published CAV reference strains. Sequence data indicated that there are many motifs in the non-coding area of about 400bp as the binding sites for transcriptional factors. All these motifs were very conservative. There were some mutations in 3 genes VP1, VP2 and VP3. Relatively, VP1 was less conservative than VP2 and VP3. Among different strains, mutations in these 3 genes were not correlated.

[Influence of avian reovirus infection on the Bursa and immune-reactions in chickens].

The influences of avian reovirus (ARV) infection at 1 day of age on bursa development, antibody responses after vaccinations to avian influenza virus (AIV), Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV), and pathogenecity of virulent IBDV (v-IBDV) were studied in chickens of SPF-origin. The results indicated that LY strain ARV infection in 1-day-old chickens caused atrophy of the Bursa and decreased lymphocyte numbers in the bursa, but it gave no significant negative effects on growth rates and antibody titers to AIV and NDV after vaccination. LY strain ARV infection decreased antibody titers to IBDV in B87 vaccinated birds but all vaccinated birds infected with ARV were still full protected from death or clinical syndromes after v-IBDV challenge. Although all B87-vaccinated birds were full protected from death after v-IBDV challenges, the antibody titers to AIV or NDV after vaccinations with inactivated vaccines were significantly lower in v-IBDV challenged birds than controls. Supprisingly, ARV infection at ages of 1-7 days could compromise the immune suppression induced by v-IBDV in B87-vaccinated birds as HI antibody titers to AIV and NDV in ARV-infected groups were significantly higher than chicknes with no ARV infection. A discussion was made on the interactions between ARV infection and vaccine IBDV or v-IBDV to explain such sophisticated phenomena in the bird experiments.

[Sequence analysis of integration sites of reticuloendotheliosis virus LTR in fowlpox vaccine virus genomes].

By use of genomic DNA prepared from 5 fowlpox virus (FPV) vaccines made in China (from Shandong, Beijing, Liaoning, Zhejing and Shanghai respectively) as the templates, reticuloendotheliosis virus (REV) LTR was amplified in PCR with a pair of primers synthesized according to the sequences flanking the integrated REV-5'LTR in FPV genomes published in US and Australia. Sequence analysis indicated that the REV-5'LTR integration sites in genomes of all 5 Chinese PFV vaccine products were identical to American and Australian FPV vaccines with integrated REV-5' LTR. Among the 5 FPV vaccine products made in China, 3 productes Vac-B-Ch, Vac-D-Ch and Vac-E-Ch had the integrated REV-5' LTR sequences of 223bp with 100% homology to that in American vaccine Vac-3-Am and Australian vaccine Vac-M3-Au. The integrated 505bp REV-LTR sequences in another Chinese products Vac-A-Ch and Vac-C-Ch had 99.6% homology to the integrated REV-LTR of American vaccine Vac-1-Am and Australian vaccine Vac-S-Au. However, REV-5'LTR integrated in all 5 FPV vaccines made in China gave only 75.4%-91.5% homology to LTR of a Chinese field strain HA9901 of REV. Based on the above results, it is reasonable to speculate that the virus stocks for the 5 Chinese FPV vaccine products were rather originally imported with their integrated REV LTR than recombinated with LTR of REV local field strains in China.

[Newcastle disease virus heredity mutation and correlation of HN and F gene].

Prevailed Newcastle disease virus isolates were collected during 1999-2005 in China. These isolates were purified by CEF plaque assay and replicated in SPF embryos. The fusion protein (F) gene and hemagglutinin-neuraminidase (HN) gene of these isolated viruses were cloned and sequenced. Some of the F gene and HN gene sequences from GenBank were also used in this study. The homologies of nucleotide and amino acids were compared and correlations were analyzed by SPSS8.0 software among different length sequences of the F gene or HN gene. The nucleotide homologies and correlation among the F gene and HN gene were also analyzed. The results indicated there are good correlation among different length sequences of the F gene or HN gene and the F genome or HN genome (r > or = 0.973). There was also good correlations among different length amino acids of NDV F protein or HN protein (0.911< or = r < or = 0.968). But, there was only a less correlation between the whole F gene and HN gene (r = 0.312). The heredity mutation of HN genes had the character of geographical areas. The sequences of HN gene in Chinese isolates had an identity of more than 97%. But there was only 79.2% - 80.7% in HN nucleotide homology among the Chinese isolates and La Sota (vaccine).

[Molecular characterization of a genotype II virulent Newcastle disease virus isolate from chicken].

Newcastle disease virus (NDV) field strain SQZ04 was isolated from a broiler flock with typical symptoms and lesions, and cloned by plaque-purification three times. NDV SQZ04 was determined as a virulent strain with MDT of 50.5h and 51.2h, ICPI of 2.0 and 1.92, IVPI of 2.8 and 2.68 respectively before and after plaque-purification. Analysis of F gene indicated that SQZO4 was determined as a virulent gene type II , and its protein amino acid sequence has homologies of 99.3% , 98.7% and 96.9% with published gene type II vaccine strains LaSota, B1, virulent strain Taxas48,much higher than homologies of 88.3% - 88.6% or 91.3% - 92.1% with published gene types VI and IX. This is the first virulent field strain of gene type UI reported in China. Further more, the amino acid sequence 111 GGRQGRL117 in the F protein cleavage site in SQZ04 strain is identical to lentogenic strains of NDV, such as vaccine strains LaSota, B1. This is the first report that virulent NDV could have lentogenic amino acid sequence in the cleavage site of F protein, where HN genes was compared SQZ04 has a higher homologies of 95.3%- 97.3% with known velogenic strains, but lower homologies of 87.8% - 89.5% with published lentogenic strains.

[Immunogenicity of envelope glycoprotein gene of reticuloendotheliosis virus expressed in insect cell].

A recombinant baculovirus expressing reticuloendotheliosis virus env gene was constructed with Bac-to-Bac Baculovirus Expression Systems. After transfecting the recombinant virus into Sf9 cells for 3 days, REV env can be detected by indirect immunofluorescence antibody assay (IFA) and Western blot with specific monoclonal antibodies of REV. The oil-water emulsion vaccine was then produced using this infected Sf9 cells lycates and inoculated SPF chickens to validate the immunogenicity for REV. The results show that special anti-REV antibody can maintain more than 45 days and resist the infection of REV viruses. This is the first success to induce anti-REV antibody in chickens by none-live viruses.

[Study on the self-assembly ability of expressed capsid protein of rabbit haemorrhagic disease virus that fused with foreign epitope at the N-terminus].

The RHDV capsid protein (VP60) gene was first subcloned into the transfer plasmid pBLUEBACHIS2B located downstream of a 6 * HIS tag, then the recombinant transfer plasmid DNA were cotransfected Sf 9 cells with Bac-N-Blue DNA and purified for cloned recombinant baculovirus by plaque assay. The expression of fused-VP60 gene was analyzed by SDS-PAGE and Western blot. A specific 69 kD protein band was obtained. Observed under electron micrography, the recombinant baculovirus expressed VP60 protein assembled into viruslike particles which were morphologically and antigenically similar to native RHD virus but did not package RNA. The close-to-native conformation of the VLPs was also supported by the haemagglutination test, in which recombinant VLPs, like RHDV, agglutinated human blood type O erythrocytes.

[Study on the characterization of the bi-directional promoter between pp38 gene and 1.8kb mRNA transcripts of Marek's disease viruses].

The bi-directional promoter between pp38 gene and 1.8kb mRNA transcripts of Marek's disease viruses (MDV) was divided into two single-direction promoters from the replication of MDV genomic DNA. The pp38 gene was cloned into pUC18 vector for plasmid pUC-pp38. Then the complete bi-directional promoter was cloned into pUC-pp38 in two directions to form plasmids pPro(f)pp38 and pPro(r)pp38, and the divided two single directional promoters were cloned in pUC-pp38 for plasmids pdPro(f)pp38 and pdPro(r)pp38. 24 to 48 hours after transfection to chicken embryo fibroblast (CEF) cells, the expression of pp38 could be detected in above 4 samples with Indirect Immuno-fluorescent Assay (IFA). In order to analysis the activity of the promoter quantificationally, CAT was used as the report gene. The complete or divided promoters were cloned into pCAT-Basic vector for plasmids pPro(f)CAT, pPro(r)CAT, pdPro(f)CAT and pdPro(r)CAT. The activity of CAT was measured from the lysed CEF cells, when they were transfected for 48 hours by the above four plasmids, respectively. The results showed the activity of the divided promoters reduce on both directions, especially for the direction of 1.8kb mRNA transcript, nearly down to 1/41.

[PCR detection of PCV-2 and PRRSV in porcine pleuropneumonia samples].

PCV-2 (Porcine circovirus type-2, PCV-2) and PRRSV (Porcine reproductive and respiratory syndrome virus, PRRSV) were detected by PCR from 253 porcine pleuropneumonia samples and 125 clinically healthy lung samples were collected from different districts of Shandong province. The results showed that 171 samples for PCV-2 and 101 samples for PRRSV were positive. The positive ratio were 67.5% and 40%, respectively. The co-infection number of PCV-2 and PRRSV was 68 in 253 samples, the positive ratio was 26.8%. While in the clinically healthy samples, only 21 samples for PCV-2 and 12 samples for PRRSV were detected positive, the positive ratio were 16.8% and 9.6%, respectively, no co-infection samples were found. Statistical result showed significant difference between positive ratio for PCV-2 and PRRSV in porcine pleuropneumonia samples and that of in clinically healthy samples. The above results demonstrated that there maybe some relationship between the infection of porcine pleuropneumonia and PCV-2 and PRRSV in pigs.

[Construction of infectious clone of subgroup J avian leukosis virus strain NX0101 and its pathogenicity].

By using PCR, 3 fragments of provirus cDNA of avian leukosis virus (ALV-J) strain NXO101 were amplified from the genomic DNA of ALV-J infected cells,and then combined in the right direction and sequences into recombinant plasmid pALV-J-NX, containing the whole genome of NX0101. After transfection of chicken embryo fibroblast (CEF) cells with plasmid pALV-J-NX DNA, the rescued virus was identified in CEF by indirect fluorescence antibody test with ALV-J specific monoclonal antibody JE9. The rescued virus could replicate in CEF at a titer of 10(5.6)/mL. The chicken experiment demonstrated that the rescued virus was still able to induce tumors in commercial meat-type broilers.

[Emerging of avian leukosis virus subgroup J in a flock of Chinese local breed].

Myeloid leukosis (ML) cases were first diagnosed in a chicken flock of Chinese local breed in Shan dong province. The main symptom included wasting, weight loss, anemia. It caused about 10% mortality of about 15000 birds at the age of 120-day. In the necropsy, gray-white nodules and protrusions in various sizes were commonly observed on the surface of the sternum, intestine and trachea. Almost all viscera tissues showed moderate to severe enlargement with diffuse gray-white nodules. Histological examination indicated that the tumor cells proliferated in tissues were myelocytes with eosinophilic granules in cytoplasm. In PCR with a pair of ALV-J-specific primers, 15 of 17 liver samples were positive. PCR product of one positive sample was sequenced and demonstrated 98.05% and 97.4% identity with ALV-J HPRS-103 strain at nuclei acid and amino acids level, respectively. By immunohistochemistry (IHC) technique with ALV-J monoclonal antibody, the most intense staining was in the tumor tissue, liver, spleen, kidney, bone marrow, and proventriculus. The results indicate that ALV-J already caused chickens infection and dead in Chinese local breed.

Virus-encoded miR-155 ortholog is an important potential regulator but not essential for the development of lymphomas induced by very virulent Marek's disease virus.

The microRNA (miRNA) mdv1-miR-M4, a functional miR-155 ortholog encoded by oncogenic Marek's disease virus (MDV), has previously been suggested to be involved in MDV pathogenesis. Using the technique of bacterial artificial chromosome mutagenesis, we have presently evaluated the potential role of mdv1-miR-M4 in the oncogenesis of the very virulent (vv) MDV strain GX0101. Unexpectedly, deletions of the Meq-cluster or mdv1-miR-M4 alone from the viral genome strongly decreased rather than abolished its oncogenicity. Compared to GX0101, mortalities of mutants GXΔmiR-M4 and GXΔMeq-miRs were reduced from 100% to 18% and 4%, coupled with the gross tumor incidence reduction from 28% to 22% and 8%, respectively. Our data suggests that the mdv1-miR-M4 is possibly an important regulator in the development of Marek's disease (MD) lymphomas but is not essential for the oncogenicity of vvMDV. In addition, some of the other Meq-clustered miRNAs may also play potentially critical roles in vvMDV induction of lymphomas.

[Identification of a new subgroup of avian leukosis virus isolated from Chinese indigenous chicken breeds].

In order to clarify Avian leukosis virus (ALV) characteristics from Chinese native chicken breeds, three ALV JS11C1, JS11C2 and JS11C3 were isolated from Chinese native breed "luhua" by inoculation of DF1 cell culture and detection of p27 antigen. Using PCR amplification of env gene, the amplified gp85 genes were analyzed and compared to all six chicken ALV subgroups reported. The gp85 genes of these three viruses were 1 005bp in length and encoded 335 amino acids, and the gp37 genes were 609bp and encoded 203 amino acids. The homology of gp85 among these three isolated strains was 91.9%-97.0%. Comparing to 18 stains of subgroup A, B, C, D, E published in GenBank, the homology was only in the range of 77.7%-84.6%, significantly lower than the gp85 homology observed within the common chicken subgroups A (88.2%-98.5%), B (91.6%-98.8%), and E (97.9%-99.4%). The gp85 homology compared with subgroup J was only 34.2%-36.5%. These results suggested that three isolated strains from Chinese native breed "luhua" belong to a new subgroup different from all six known subgroups from Chickens, and thus designated as subgroup K.

[The ALV-A/B specific antibodies correlation between ELISA and IFA detection in chicken serum].

To study the correlation between ELISA and IFA tests in detection of ALV-A/B antibody in chicken sera, ELSA S/P values and IFA titers for different serum samples were measured and statistically analyzed. The results indicated that there was a strong positive correlation between ELISA S/P values and IFA titers (r = 0.97435, P < 0.001). Because the positive correlation between ELISA and IFA was so strong and antibody positive rates were identical in two tests, it suggested that IFA could be used as a alternative method to replace ELISA kit when only limited numbers of samples to be tested to reduce the cost and increase the sensitivity.