To develop the polymerase chain reaction (PCR) for the detection of type D simian retrovirus (SRV) infection, an oligonucleotide primer pair was designed to hybridize to the sequences within euv gene of SRV subtype 1 (SRV-1). The 3'proximal env sequences annealing to the primers had been rather conserved among three different subtypes of SRV, SRV-1, SRV-2, and SRV-3 (Mason-Pfizer Monkey Virus: MPMV). The PCR using the primer pair targetingan an env region the successfully detected and amplified all three subtypes of SRV with excellent specificity after single round of reaction. The tests with peripheral blood mononuclear cells infected either with simian immunodeficiency virus or simian T-lymphotropic virus type 1, maior immunosuppressive viral agents together with SRV in simian, verified the specificity of the PCR by excluding any cross reactivity. Semiquantitative titration PCR, amplifying serially diluted plasmid DNA of each subtype, was performed to evaluate sensitivity limits of the reaction. Based on molecular weight of each cloned SRV genome, the PCR should be able to detect one SRV-infected cell per more than 5-7x104 uninfected cells after simple ethidium bromide staining of resulting products. The PCR must be very efficient screening sisters with its quickness, certainty, and sensitivity for SRV-infected animals used in human AIDS research model. Second round amplification of the reaction products from the first PCR, or Southern hybridization by radiolabeled probes shall render to compete its efficacy to ELISA which has been the most sensitive technique to screen SRV infection but with frequent ambiguity problem.
Animals ; Betaretrovirus* ; Clone Cells ; DNA* ; Enzyme-Linked Immunosorbent Assay ; Ethidium ; Genome ; Haplorhini ; Humans ; Mason-Pfizer monkey virus ; Mass Screening ; Molecular Weight ; Plasmids ; Polymerase Chain Reaction* ; Retroviruses, Simian ; Sensitivity and Specificity ; Siblings ; Simian Immunodeficiency Virus

This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.
Amino Acid Sequence ; Animals ; Betaretrovirus ; chemistry ; classification ; genetics ; physiology ; Cell Transformation, Viral ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Molecular Sequence Data ; NIH 3T3 Cells ; Phylogeny ; Retroviridae Infections ; veterinary ; virology ; Sequence Alignment ; Sheep ; Sheep Diseases ; virology ; Transformation, Genetic ; Tumor Virus Infections ; veterinary ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

To investigate the kinship between the Inner Mongolia pandemic strain and representative strains of the Jaagsiekte sheep retrovirus (JSRV), total DNA from the lung tissue of a JSRV-infected sheep in Inner Mongolia was used to clone fragments of gag, pro and pol genes. The recombinant plasmid pMD-JSRV (including complete genomic sequence of the JSRV strain isolated from Inner Mongolia) was constructed by linking all the cloned fragments with long terminal repeat (LTR) and env gene fragments (cloned previous and reserved by our research team). Sequence analyses revealed that the genome was 7690 bp in length and contained several typical molecular markers for exogenous form of JSRV. These included the Sca I restriction site in the gag gene, two predicted "CCHC" motifs of zinc finger in the encoded nucleocapsid protein and the predicted "YXXM" motif in the TM region of Env. Homology analyses showed that the virus strain belonged to the JSRV type II. pMD-JSRV and AF105220 strains shared a nucleotide identification of 95%. The full length genomic clone of JSRV could provide a molecular basis for an infectious JSRV molecular clone as well as an experimental platform to study the detection and pathogenesis of JSRV.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Genome, Viral ; Jaagsiekte sheep retrovirus ; genetics ; Pandemics ; Plasmids ; Proviruses ; genetics

OBJECTIVETo generate two genetically engineered mouse models of ErbB2/Neu positive-PTEN deficient breast cancer and to compare their biological properties.

METHODSThe genetically engineered mice previously developed with mouse mammary tumor virus (MMTV) promoter driven expression of activated ErbB2/Neu and recombinant Cre (FVB/N-MMTV-NIC) were interbred with Flox-PTEN mice; and FVB/N-ErbB2KI mice, harboring endogenous promoter driven activated ErbB2/Neu expression, FVB/N-MMTV-Cre mice and the flox-PTEN mice were interbred. Neu, Cre and PTEN genes were amplified by PCR for genotyping of the offsprings. ErbB2/Neu and PTEN expression in mammary tumors were detected by immunohistochemistry. Tumor formation time, tumor number, histopathology and lung metastasis were compared between two models, Ki-67 expression was detected by immunohistochemistry, and TUNEL staining of tumor tissues was performed.

RESULTSTwo genetically engineered mouse models of ErbB2/Neu positive-PTEN homozygous deficient breast cancer were generated. The models were confirmed by genotyping and immunohistochemistry. One model with exogenous MMTV promoter driven expression of activated ErbB2/Neu and Cre coupling PTEN disruption was designated as NIC/PTEN(-/-) mice, and the other with MMTV-Cre induced endogenous promoter driven expression of activated ErbB2/Neu with PTEN disruption was designated as ErbB2KI/PTEN(-/-) mice. The tumor formation time in NIC/PTEN(-/-) mice was significantly shorter than that of ErbB2KI/PTEN(-/-) mice (30 vs 368 d, P<0.01); the number of tumor and incidence of lung metastasis was also significantly higher in NIC/PTEN(-/-) mice (10 vs 1-2 and 75.0% vs 37.5%, respectively, Ps<0.01). The Two models displayed distinct histopathological morphology. NIC/PTEN(-/-) tumor showed more Ki-67 positive cells than ErbB2KI/PTEN(-/-) tumor did (86.9%±2.8% vs 37.4%±7.2%, P<0.01), while the amount of cell apoptosis in tumors was not significantly different between two models.

CONCLUSIONTwo genetically engineered mouse models of ErbB2/Neu positive-PTEN homozygous deficient breast cancer with different phenotypes have been successfully generated, which may provide useful resource for further investigation of the initiation and progression of HER2/ErbB2 breast cancer, as well as for the development of novel prevention and treatment regimens of this malignance.

Animals ; Breast Neoplasms ; genetics ; Disease Models, Animal ; Female ; Gene Deletion ; Mammary Neoplasms, Animal ; Mammary Tumor Virus, Mouse ; genetics ; Mice ; Mice, Transgenic ; PTEN Phosphohydrolase ; genetics ; Receptor, ErbB-2 ; genetics

To carry out pathologic diagnoses and whole-genome sequence analyses of the Jaagsiekte sheep retrovirus (JSRV) in Xinjiang, China, we first observed sheep suspected to have the JSRV. Then, the extracted virus suspension was observed by transmission electron microscopy (TEM). Total RNAs from lungs of JSRV-infected sheep were extracted and reverse-transcribed using a cDNA synthesis kit. Six pairs of primers were designed according to the exogenous reference virus strain (AF105220). Reverse transcription-polymerase chain reaction was carried out from JSRV-infected tissue, and the whole genome of the JSRV sequenced. Our results showed: flow of nasal fluid ("wheelbarrow test"); different sizes of adenoma lesions in the lungs; papillary hyperplasia of alveolar epithelial cells; alveolar cavity filled with macrophages; dissolute nuclei in central lesions. TEM revealed JSRV particles with a diameter of 88 nm to 125. 4 nm. The full-length of the viral genome sequence was 7456 bp. BLAST analyses showed nucleotide homology of 96% and 95% compared with that of the representative strain from the USA (AF105220) and UK (AF357971). Nucleotide homology was 89.8% and 89.9% compared with the endogenous Jaagsiekte sheep retrovirus, Inner Mongolia strain (DQ838493) and USA strain (EF680300). The specific pathogenic amino-acid sequence "YXXM" was found in the TM district, similar to the exogenous JSRV: this gene has been reported to be oncogenic. This is the first report of the complete genomic sequence of the exogenous JSRV from Xinjiang, and could lay the foundation for study of the biological characteristics and pathogenic mechanisms of the pulmonary adenomatosis virus in sheep.
Amino Acid Sequence ; Animals ; China ; Genome, Viral ; Jaagsiekte sheep retrovirus ; classification ; genetics ; isolation & purification ; pathogenicity ; Lung ; pathology ; virology ; Molecular Sequence Data ; Phylogeny ; Pulmonary Adenomatosis, Ovine ; pathology ; virology ; Sheep ; Viral Proteins ; chemistry ; genetics ; Virulence

PURPOSE: We investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) could promote the development of preinvasive and invasive breast cancer in mouse mammary tumor virus (MMTV-erbB2) mice with estrogen receptor-positive tumors. METHODS: MMTV-erbB2 mice were randomly divided into three experimental groups with 20 mice in each group. MMTV-erbB2 mice were treated with daily subcutaneous injections of vehicle or rhG-CSF (low-rhG-CSF group, rhG-CSF 0.125 microg; vehicle-rhG-CSF group, normal saline 0.25 microg; and high-rhG-CSF group, rhG-CSF 0.25 microg) at 3 months of age. Cellular and molecular mechanisms of G-CSF action in mammary glands were investigated via immunohistochemistry and reverse transcription polymerase chain reaction. RESULTS: Low, but not high, rhG-CSF doses significantly accelerated mammary tumorigenesis in MMTV-erbB2 mice. Short-term treatment with rhG-CSF could significantly promote the development of preinvasive mammary lesions. The cancer prevention effect was associated with reduced expression of proliferating cell nuclear antigen, cluster of differentiation 34, and signal transducers and activators of transcription 3 in mammary glands by >80%. CONCLUSION: We found that G-CSF was regulated by rhG-CSF both in vitro and in vivo. Identification of G-CSF genes helped us further understand the mechanism by which G-CSF promotes cancer. Low doses of rhG-CSF could significantly increase tumor latency and increase tumor multiplicity and burden. Moreover, rhG-CSF effectively promotes development of both malignant and premalignant mammary lesions in MMTV-erbB2 mice.
Animals ; Breast Neoplasms ; Carcinogenesis ; Cell Proliferation ; Estrogens* ; Granulocyte Colony-Stimulating Factor* ; Humans ; Immunohistochemistry ; Injections, Subcutaneous ; Mammary Glands, Human ; Mammary Tumor Virus, Mouse ; Mice* ; Polymerase Chain Reaction ; Proliferating Cell Nuclear Antigen ; Reverse Transcription ; Transducers

Ovine pulmonary adenomatosis (OPA) is a naturally occurring contagious lung tumor of sheep which was caused by an exogenous retrovirus of sheep, jaagsiekte retrovirus (JSRV). Although no specific circulating antibodies against the virus coud be detected in infected sheep, exogenous JSRV proviral DNA sequences (exJSRV) and JSRV RNA transcripts could be detected in lung tumors, lymphoreticular system and peripheral blood mononuclear cells (PBMC) from sheep affected by OPA. The sheep genome carried 15 to 20 copies of endogenous retrovirus loci (enJSRV) that were similar to JSRV in structural genes but the divergene in U3. Therefore, primers specific for the U3 sequences of exJSRV were designed for the specific PCR and nested PCR (n-PCR). Sensitivity between specific PCR assay and n-PCR assay was compared by using serial dilutions of positive plasmid pJSRV-LTR in a background of 700ng sheep genome DNA. Sensitivity of n-PCR was ten-fold higher than specific PCR. The n-PCR was only available in blood test for detection of JSRV infected sheep and might be useful in epidemiological studies and disease control of OPA.
Animals ; Base Sequence ; Clinical Laboratory Techniques ; DNA, Viral ; analysis ; Endogenous Retroviruses ; genetics ; Jaagsiekte sheep retrovirus ; genetics ; Lung Neoplasms ; virology ; Molecular Sequence Data ; Polymerase Chain Reaction ; Pulmonary Adenomatosis, Ovine ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity ; Sequence Analysis, DNA ; Sheep ; Sheep Diseases ; virology ; Virus Cultivation

OBJECTIVETo develop a safe and effective lentivirus vaccine model and provide insights into the development of other lentivirus vaccines.

METHODSIn this study, a construct of pGPT was made by deleting env gene in the infectious Equine infectious anemia virus (EIAV) molecular clone of WU57. Since the overlaping of EIAV Rev gene with env gene, there was no Rev gene in the construct of pGPT. For compensation of Rev function, the construct of pGPTC was made by inserting 4 copies of constitutive RNA transport elements (CTEs) from Mason-Pfizer monkey virus into the construct of pGPT. In addition, a construct designated pTEB expressing EIAV Env protein was made while env gene-minus viruses were made by co-transfection of pGPT/pTEB or pGPTC/pTEB into 293 cells. Western blot was used to identify the development of recombinant virus particles. Then immunofluorescence assay was used to evaluate the infectivity of recombinant virus particles in vitro.

RESULTSEIAV proteins expression was detected in the supernatant of transfected 293 cells by Western blot within pGPTC/pTEB transfected cells. However, no evidence of EIAV proteins expression was observed within pGPT/pTEB transfected cells. EIAV proteins expression was detected in the first round but not in the second round infected EK cells with EIAV(GPTC) by immunofluorescence assay.

CONCLUSIONRev/RRE was necessary for expression of viral structural proteins; CTEs from Mason-Pfizer monkey virus was functionally interchangeable with EIAV Rev/RRE to help RNAs transportation out of nucleus to express structural proteins and EIAV particles were produced in the transfected 293 cells. A live EIAV recombinant virus with single round infection had been developed.

Animals ; Blotting, Western ; Cells, Cultured ; Fluorescent Antibody Technique ; Genes, rev ; Haplorhini ; Horses ; Humans ; Infectious Anemia Virus, Equine ; genetics ; Lentivirus ; immunology ; Lentivirus Infections ; immunology ; prevention & control ; Mason-Pfizer monkey virus ; genetics ; Transfection ; Vaccines, Synthetic ; Viral Vaccines ; genetics ; immunology