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.
Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; classification ; genetics ; isolation & purification ; metabolism ; Breeding ; Chickens ; genetics ; virology ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Viral Envelope Proteins ; genetics ; metabolism

To study the correlation between 50% tissue-culture infective dose (TCID50) value and p27 antigen S/P value of Avian leukosis virus subgroup J and discuss their significance, chicken embryo fibroblast (CEF) cells were inoculated with Avian leukosis virus subgroup J strain NX0101 and samples were tested continuously for ten days after changing maintenance media. The correlation between TCID50 and p27 antigen S/P value of ten days were then analysized. Simultaneously, DF-1 cells were inoculated with NX0101 and passaged to 20 generations. Samples taken from 1st generation, 5th generation, 10th generation, 15th generation and 20th generation were tested for the TCID50 titer and the p27 antigen S/P value separately. A significant Pearson correlation was found between them in CEF cells (r = 0.85277; P < 0.0001) and in DF-1 cells (r = 0.93000; P = 0.0220). This study provided an important parameter for predicting TCID50 by detecting the p27 antigen S/P value.
Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; immunology ; pathogenicity ; Chick Embryo ; Fibroblasts ; virology ; Proliferating Cell Nuclear Antigen ; analysis ; immunology ; Viral Load ; immunology

The transmembrane protein (TM) encoded by gp37 gene plays a critical role when virus fusion with cell membrane occurs. Several highly conserved regions in TM are important targets for antivirus studies. Studies on structure and function of TM will provide basic information for anti-retrovirus, especially for avian leukosis virus. In the study, gp37 gene was amplified by PCR from the Chinese strain ALV-J-WS0701. The gp37 gene was cloned into pMD18-T vector, and was sequenced. Then, pFast-BacHTb-gp37 vector was constructed and expressed by baculovirus expression vector system. The expression product of gp37 gene was analyzed by indirect immunofluorescence assay and Western blot. The results showed that positive green fluorescence was present in sf9 cells infected with recombinant virus and a protein band with a molecular weight of 21kD was present in Western blot. It is concluded that gp37 gene was expressed in sf9 cells infected with recombinant virus successfully.
Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; classification ; genetics ; isolation & purification ; Cell Line ; Chickens ; Cloning, Molecular ; Gene Expression ; Spodoptera ; Viral Envelope Proteins ; genetics ; metabolism

Recently, avian viral diseases have become one of the main models to study mechanisms of viral infections and pathogenesis. The study of regulatory relationships and mechanisms between viruses and microRNAs has also become the focus. In this review, we briefly summarize the general situations of microRNAs encoded by avian herpesviruses. Also, we analyze the regulatory relationships between tumorigenicity of avian herpesviruses and microRNAs. Additionally, the possible applications for prevention and treatment of viral diseases (such as infectious bursal disease, avian influenza and avian leucosis) using the regulatory mechanisms of microRNAs are also discussed.
Animals ; Avian Leukosis ; Birds ; virology ; Birnaviridae Infections ; Herpesviridae ; genetics ; Influenza in Birds ; MicroRNAs ; genetics

Avian leukosis virus subgroup J (ALV-J) is an avian retrovirus that can induce myelocytomas. A high-frequency mutation in gene envelope endows ALV-J with the potential for cross-species transmission. We wished to ascertain if the ALV-J can spread across species under selection pressure in susceptible and resistant hosts. First, we inoculated (in turn) two susceptible host birds (specific pathogen-free (SPF) chickens and turkeys). Then, we inoculated three resistant hosts (pheasants, quails and ducks) to detect the viral shedding, pathologic changes, and genetic evolution of different isolates. We found that pheasants and quails were infected under the selective pressure that accumulates stepwise in different hosts, and that ducks were not infected. Infection rates for SPF chickens and turkeys were 100% (16/16), whereas those for pheasants and quails were 37.5% (6/16) and 11.1% (3/27). Infected hosts showed immune tolerance, and inflammation and tissue damage could be seen in the liver, spleen, kidneys and cardiovascular system. Non-synonymous mutation and synonymous ratio (NS/S) analyses revealed the NS/S in hypervariable region (hr) 2 of pheasants and quails was 2.5. That finding suggested that mutation of isolates in pheasants and quails was induced by selective pressure from the resistant host, and that the hr2 region is a critical domain in cross-species transmission of ALV-J. Sequencing showed that ALV-J isolates from turkeys, pheasants and quails had moved away from the original virus, and were closer to the ALV-J prototype strain HPRS-103. However, the HPRS-103 strain cannot infect pheasants and quails, so further studies are needed.
Amino Acid Sequence ; Animals ; Avian Leukosis ; transmission ; virology ; Avian Leukosis Virus ; classification ; genetics ; physiology ; Chickens ; Ducks ; virology ; Galliformes ; virology ; Host Specificity ; Molecular Sequence Data ; Poultry Diseases ; transmission ; virology ; Quail ; virology ; Sequence Alignment ; Turkeys ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

Two strains of Avian leukosis virus subgroup B (ALV-B) were isolated for the first time in China Hy-line White on the cultured DF-1 cells which were inoculated tissue samples from by an ELISA assay, a histopathology examination and a PCR-based diagnosis. The results from the ELISA assay indicated that the positive rate of serum antibodies to ALV-B and ALV-J virus were 16.3% (15/92) and 13% (12/92), respectively. The histopathological examination indicated that two types of tumor cells existed at same focus in liver and spleen, which mainly were myelocytoma cells and lymphosarcoma cells. The PCR-based diagnosis were performed as follows: the cellular DNA was extracted from the inoculated DF-1 cells; the specific fragments of 1100 bp and 924 bp were obtained by a PCR system with the diagnostic primers of ALV-B and ALV-J; and the PCR results for ALV-A, MDV and REV were all negative. Then, the amplified fragments of the two ALV-B stains were partially sequenced and shown an identity of 92.8%,94.7% with the prototype strain of ALV-B (RSV Schmidt-ruppin B). The identities of two ALV-J strains with the prototype strain HPRS-103 at 96.9%, 91.5%; The identities of two ALV-J strains with the American prototype strain at 85.9%, 81.5%. Our study had shown that ALV-B was isolated for the first time from the ALV-J infected commercial layer flocks in China. It also indicated that the chance of genetic recombination among various subgroups of ALV was increased.
Animals ; Avian Leukosis ; pathology ; virology ; Avian Leukosis Virus ; classification ; genetics ; isolation & purification ; Cell Line ; Chickens ; China ; Liver ; pathology ; virology ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; pathology ; virology ; Spleen ; pathology ; virology

Two subgroup J avian leukosis viurses (ALVs) were isolated from broiler breeder flocks, in which myeloid leukosis had occurred. The isolates could be classified as subgroup J ALV. by the positive reaction in polymerase chain reaction (PCR) with primers specific for subgroup J ALV. Two isolates replicated in chicken embryo fibroblast (CEF) cells from the alv6 chicken line in which cells are resistant to subgroup A and E ALVs. In in vitro serum neutralization tests with other subgroup ALVs including ADOL-Hc1, the prototype of subgroup J ALVs isolated in the United States of America, two isolates were partially neutralized by antibody to ADOL-Hc1, indicating that Korean isolates and ADOL-Hc1 may be antigenically related, but not identical. When the PCR was done with a primer pair designed to amplify genes of E element and long terminal repeat of proviral DNA, the PCR product size of one isolate (KOAL-PET) was smaller than that of ADOL-Hc1, suggesting that some sequences in these regions are deleted.
Animals ; Antibodies, Viral/immunology ; Antigens, Viral/immunology ; Avian Leukosis/virology ; Avian leukosis virus/*classification/genetics/immunology/*isolation & purification ; Cell Line ; Chick Embryo ; Chickens/*virology ; Korea ; Neutralization Tests ; Polymerase Chain Reaction ; Poultry Diseases/virology

Abstract:Subgroup J avian leukosis virus (ALV-J) infect cells by binding to the chNHE1 receptor protein of the host and causes tumors. The tumor incidence of the ALV-J-infected chickens was observed by histo pathology, and virus was isolated on DF-1 cell line. The ALV-J load and mRNA of chNHElreceptor protein were detected by real time PCR. The relationship between ALV-J load, chNHE1 receptor expression levels and tumor spectrum was analyzed. The results showed that the tumors induced by ALV-J in laying hens and local lines of chicken were different. No significant relationship was observed between ALV-J load and tumor spectrum. ALV-J load was positively correlated with mRNA expression of chNHE1. The mRNA expression of chNHE1 increased when the tumors occurred. Our results suggest the chNHE1 protein is not only the receptor of ALV-J infected host but also play an important role in the process of tumor development. This study provides a scientific basis for further studying of oncogenic mechanism of ALV-J.
Animals ; Avian Leukosis ; genetics ; metabolism ; virology ; Avian Leukosis Virus ; genetics ; physiology ; Chickens ; genetics ; metabolism ; Poultry Diseases ; genetics ; metabolism ; virology ; Receptors, Virus ; genetics ; metabolism ; Sodium-Hydrogen Exchangers ; genetics ; metabolism ; Viral Load

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.
Animals ; Antibodies, Viral ; blood ; immunology ; Avian Leukosis ; diagnosis ; immunology ; virology ; Avian Leukosis Virus ; classification ; immunology ; isolation & purification ; Cell Line ; Chickens ; Enzyme-Linked Immunosorbent Assay ; methods ; Fluorescent Antibody Technique, Indirect ; methods ; Poultry Diseases ; diagnosis ; immunology ; virology ; Species Specificity

By inoculation of blood samples in DF-1 (C/E) cell culture, an exogenous avian leukosis virus (ALV) strain SDAU09C2 was isolated from a breeder farm of Chinese native breed "Luhua" in Shandong province. Comparisons of the amino acid sequence of env gene gp85 from the isolate with those from other ALV reference strains of different subgroups indicated that SDAU09C2 had the highest gp85 identity to two reference strains of subgroup B of 92.5%. Its gp85 identity to other chicken ALV subgroups A, C, D, E was in the range of 73.2%-87.9%. The identity to subgroup J was only 30.3%-32.4%. This is the first report on isolation and identification of ALV-B and its gp85 from Chinese native breed chickens.
Amino Acid Sequence ; Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; chemistry ; classification ; genetics ; isolation & purification ; Breeding ; Chickens ; Female ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Viral Envelope Proteins ; chemistry ; genetics