禽白血病病毒J亚群env基因产物的抗原性分析

用PCR扩增方法将ALV Jenv基因不同片段进行了克隆 ,并构建了env基因片段GST融合蛋白载体。用Westernblot实验证明 ,大肠杆菌表达的不同env基因片段的GST融合蛋白能与相应的单克隆抗体产生特异性反应性 ,单克隆抗体JE9和G2识别的抗原位点位于gp85的氨基酸 6 5～ 1 5 5区域 ,而I45识别的抗原表位位于env基因的另一区域 (1 5 6～ 2 3 3位氨基酸 )。ALV J氨基酸多肽而非糖基化位点决定ALV J的亚群特异性     

Characterization of Determinants for Envelope Binding and Infection in Tva, the Subgroup A Avian Sarcoma and Leukosis Virus Receptor

Tva is the cellular receptor for subgroup A avian leukosis and sarcoma virus (ALSV-A). The viral interaction domain of Tva is determined by a 40-residue, cysteine-rich module closely related to the ligand binding domain of the human low-density lipoprotein receptor (LDLR). In this report, we examined the role of the LDLR-like module of Tva in envelope binding and viral infection by mutational analysis. We found that the entire LDLR module in Tva is essential for efficient binding to the viral envelope protein. However, the 17 N-terminal residues of this module can be deleted without affecting receptor function, suggesting that the major determinants for viral entry are located at the C terminus of the module. The effect on viral infection of many amino acid substitutions and deletions in the LDLR module is context dependent, suggesting that the residues important for viral entry are dispersed throughout the LDLR module. In addition, we found that all 27 mutations at residues D46, E47, and W48 greatly reduced envelope binding. These results are discussed in relation to a recently elucidated structure for an LDLR module.     

禽白血病病毒J亚群env基因的克隆和序列分析

应用多聚酶链反应（PCR）的方法扩增出ADOL-4817毒株的囊膜蛋白env基因,并克隆进大肠杆菌。经核酸序列分析证明,env基因的大小为1746 bp,其中gp85和gp37由1554 bp组成,可翻译成517个氨基酸,分子量为57.7 D。根据糖基化位点N-X-S/T的特点,发现ADOL-4817的env蛋白有15个潜在的糖基化位点。同源性分析证明,ADOL-4817的env基因与其它ALV-J的env基因序列同源性为88.8%～92.4%,而与外源性ALVs的相应序列的同源性仅为40.5%～51.4%,然而,与内源性的EAV-HP毒株的类env基因的同源性高达91.2%;另外,ADOL-4817毒株的gp37在C末端多了13个氨基酸。这些结果提示,ALV-J的env基因存在广泛的变异性,env基因可能来源于内源性和外源性ALVs的重组。     

禽白血病病毒J亚群囊膜蛋白env基因的克隆和表达

禽白血病病毒J亚群（ALV－J）是90年代鉴定出的ALV的新亚群,其囊膜蛋白env基因序列别与ALV A－E亚群的有相当大的差别。为ALV－J env基因及春表达产物的特点,用PCR方法扩增出ADOL－4817毒株的env基因,并克隆进TA载体,经电泳鉴定大小为1.7kb。将克隆出的env基因与杆状病毒pBlue-Bac4表达质粒DNA连接,构建成转移性载体pBac4817env,通过与Bac-N-Blue杆状病毒DNA共转染,区得了重组病毒rBac4817env-2。该重组杆状病毒感染Sf9细胞,能高效表达env基因产物,免疫荧光分析结果证明,单克隆抗体G2或多价兔抗env gp37血清能识别Sf9细胞,能高效表达env基因表达的特异性抗原;Western blotting分析结果表明,表达的重组基因产物的分子量大小约为90kD-94kD。用这些重组基因产物免疫鸡可以诱导鸡导鸡产生出高滴度的抗ALV－J特异性抗体。这一结果提示,这种杆状病毒表达的重组基因产物有助于ALV－J env基因生物学特性的深入研究。     

禽白血病病毒J亚群env基因的克隆和序列分析

应用多聚酶链反应（PCR）的方法增出ADOL－4817毒株的囊膜蛋白env基因,并克隆进大肠杆菌。经核酸序列分析证明,env基因的大小为1746bp,其中gp85和gp37mh 1554bp组成,可翻译成517个氨基酸,分子量为57.7kD。根据糖基化位点N－X－S／T的特点,发现ADOL－4817的env蛋白有15个潜在的糖基化位点。同源性分析证明,ADOL－4817的env基因与其它ALV－J的env基因序列同源性为88.8％－92.4%,而与外源性ALVs的相应序列的同源性仅为40.5％－51.4％,然而,与内源性的EAV－HP毒株的类env基因的同源性高达91.2％;另外,ADOL－4817毒株的gp37d C末端多了13个氨基酸,这些结果提示,ALV－J的env基因存在广泛的变异性,env基因可能来源于内源性和外源性ALVs的重组。     

A Charged Second-Site Mutation in the Fusion Peptide Rescues Replication of a Mutant Avian Sarcoma and Leukosis Virus Lacking Critical Cysteine Residues Flanking the Internal Fusion Domain

The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.All retroviruses have envelope glycoproteins that interact with a receptor protein on the cell surface to initiate entry (18, 36). The viral glycoprotein is synthesized as a precursor polyprotein consisting of the surface (SU) glycoprotein, which contains the domains that bind with the cellular receptor, and the transmembrane (TM) glycoprotein, which tethers the protein to the viral surface and contains the domains responsible for fusion of the viral and cellular membranes (32). After synthesis, the precursor viral glycoproteins form trimers through the interaction of the TM domains. The SU and TM domains are then cleaved by a cellular protease, forming a mature, metastable complex capable of mediating viral entry. A specific receptor protein interaction with the SU domain of the mature Env is required to initiate a conformational change in the trimer, separating the globular SU domains to allow the TM glycoproteins to form a structure that projects the fusion peptide toward the target membrane. Two domains in TM, the N-terminal heptad repeat and the C-terminal heptad repeat, are critical for the formation of the extended structure (13, 31)}. The fusion peptide is thought to interact with a target membrane irreversibly, forming an extended prehairpin TM oligomer structure anchored in both the viral and target membranes (35). The cooperation of several of these extended prehairpin TM oligomer structures is most likely required to complete fusion. The viral and target membranes are brought into close proximity when the C-terminal heptad repeats fold back into grooves formed by the N-terminal heptad repeats, forming presumably the most stable TM structure, the six-helix bundle (6HB). Fusion of the membranes proceeds through the initial mixing of the outer lipid leaflets, hemifusion, followed by initial fusion pore formation, pore widening, and the completion of fusion. The 6HB may undergo some additional structural rearrangement in order to bring the fusion peptide and membrane-spanning domain of TM into close proximity to form the final trimeric hairpin structure (22, 24, 33).Until recently, the triggering of class I virus fusion proteins was thought to occur by one of two mechanisms (13, 35, 36). In one mechanism, the viral glycoproteins interact with receptors on the cell surface, resulting in the trafficking of the virion into an endocytic compartment, followed by the triggering of structural rearrangements in the viral glycoproteins to initiate fusion by exposure to low pH (e.g., influenza virus hemagglutinin [HA]). In a second entry mechanism, the interaction of the viral glycoproteins with receptors on the cell surface in a neutral pH environment triggers the structural rearrangements in the viral glycoproteins directly, initiating viral entry. Retroviruses predominately employ the second entry mechanism, although two cellular protein receptors may be required to complete the conformational changes in the viral glycoproteins necessary to complete entry (e.g., human immunodeficiency virus type 1). However, the entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses demonstrates a third entry mechanism for the action of class I virus fusion proteins (25). ASLV entry requires both a specific interaction between the viral glycoproteins and receptors at the cell surface at neutral pH, triggering initial conformational changes in the viral glycoproteins, and a subsequent exposure to low pH to complete fusion (2, 3, 22-24).The fusion peptides of ASLVs are not at the N terminus of the cleaved TM, as in all other retroviral TM proteins, but in a proposed internal loop (TM residues 22 to 37) flanked by two cysteine residues (residues C9 and C45) (Fig. ​(Fig.1).1). The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: both contain an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond (10). Other viruses contain internal fusion peptides also predicted to be in looped structures (35). In a study to determine if the cysteines that flank the ASLV fusion peptide are required for function, mutant ASLV Env proteins were constructed with one or both of these cysteines changed to serine (C9S, C45S, or C9S C45S [C9,45S]) (8). The mutant subgroup A ASLV [ASLV(A)] Env proteins were expressed, processed, and incorporated into virions at levels similar to those of wild-type (WT) ASLV(A) Env. The mutant and WT ASLV(A) Env proteins bound the Tva receptor with similar affinities. However, murine leukemia virus (MLV) virions pseudotyped with the mutant Envs were ∼500-fold less infectious (titer, ∼2 × 10³ inclusion-forming units [IFU]/ml) than MLV virions pseudotyped with WT ASLV(A) Env (titer, ∼1 × 10⁶ IFU/ml). The ability of the mutant Envs to mediate cell fusion was also greatly impaired compared to that of WT ASLV(A) Env in a cell-cell fusion assay. We concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In a recent study, the cysteines flanking the fusion peptide region were shown to be critical for the lipid mixing stage of fusion (6).Open in a separate windowFIG. 1.Schematic representations of the ASLV-based RCASBP retroviral vector and the major domains of the envelope glycoproteins. The RCASBP(A)AP replication-competent vector contains a subgroup A env and a reporter gene coding for heat-stable AP. The hypervariable domains (vr1, vr2, hr1, hr2, and vr3) of the SU glycoprotein, the proteolytic cleavage site, the putative fusion peptide region (shaded box), and the membrane-spanning domain (MSD) of the TM glycoprotein are shown schematically. The first 45 residues of the TM glycoprotein are shown for wild-type subgroup A Env (WT) and for the three mutants tested in this study, with either a substitution of serine for the cysteine at position 9 in TM (C9S), a substitution of serine for the cysteine at position 45 in TM (C45S), or both substitutions (C9,45S). The complete sequence of the ASLV(A) WT TM glycoprotein is shown, with the fusion peptide region, N-terminal and C-terminal heptad repeat regions (N-alpha-helix; C-alpha helix), and membrane-spanning domain indicated.Very little is known about the structures of fusion peptides in the context of full-length, trimeric, viral glycoproteins upon interaction with target membranes. Also, natural membrane targets contain a variety of lipid and protein compositions in an asymmetrical organization that is difficult to reproduce experimentally (27). In addition, little is known about how fusion proteins with internal fusion peptide regions interact with target membranes or the possible conformational changes that might be required to complete the fusion process (19, 20). In this study, replication-competent ASLV(A) viruses containing the C9S, C45S, or C9,45S mutations were constructed and genetically selected for improved replication in chicken fibroblasts in order to further explore the importance of these cysteines for proper TM function. Viruses with single cysteine-to-serine mutations reverted to the WT sequence. However, viruses with both the C9S and the C45S mutation retained both mutations and acquired a second-site mutation that significantly enhanced the infectivity of the genetically selected virus population. Unexpectedly, the selected second-site mutation was a charged residue located in the middle of the hydrophobic fusion peptide within TM.     

Genetic Mapping of the Cloned Subgroup A Avian Sarcoma and Leukosis Virus Receptor Gene to the TVA Locus

A chicken gene conferring susceptibility to subgroup A avian sarcoma and leukosis viruses (ASLV-A) was recently identified by a gene transfer strategy. Classical genetic approaches had previously identified a locus, TVA, that controls susceptibility to ASLV-A. Using restriction fragment length polymorphism (RFLP) mapping in inbred susceptible (TVA*S) and resistant (TVA*R) chicken lines, we demonstrate that in 93 F₂ progeny an RFLP for the cloned receptor gene segregates with TVA. From these analyses we calculate that the cloned receptor gene lies within 5 centimorgans of TVA, making it highly probable that the cloned gene is the previously identified locus TVA. The polymorphism that distinguishes the two alleles of TVA in these inbred lines affects the encoded amino acid sequence of the region of Tva that encompasses the viral binding domain. However, analysis of the genomic sequence encoding this region of Tva in randomly bred chickens suggests that the altered virus binding domain is not the basis for genetic resistance in the chicken lines analyzed.Avian sarcoma and leukosis viruses (ASLV) may be classified into several subgroups based on properties of the viral envelope proteins. Five major subgroups of ASLV (A to E) have been defined based on immunological reactivity of the viral envelope proteins, host range, and infection interference patterns (reviewed in reference 18). The subgroup specificity of the virus maps to the env gene, and distinct hypervariable regions within env have been shown to determine the viral subgroup (2, 3, 7, 8).Patterns of susceptibility of inbred chicken lines to infection with the various ASLV subgroups suggest that three loci control viral entry for the five major ASLV subgroups (11, 12, 15, 17). Dominant alleles at the TVA and TVC (TVA*S and TVC*S) loci confer susceptibility to subgroups A and C viruses, respectively. The TVB locus is more complex, with various alleles determining infection by subgroup B, D, and E viruses. TVA, TVB, and TVC appear to act at the level of viral entry into the cell; therefore, it has been assumed that the TV loci encode or control expression of the cellular receptors for ASLV (5, 13).Recently, a gene that confers susceptibility to subgroup A ASLV (ASLV-A) was cloned by a gene transfer strategy (1, 19). Molecular clones containing coding sequences for this gene relieve the block to viral entry when expressed in a number of mammalian cell lines (1, 19). The gene encodes a surface glycoprotein that has been shown to bind directly and specifically to the ASLV-A envelope protein (4, 9). In addition, binding of the receptor protein to ASLV-A envelope protein induces conformational changes in the viral glycoproteins that appear to be associated with viral entry (10). Finally, an antibody to the receptor protein specifically blocks infection of chicken cells by subgroup A viruses, suggesting that this gene encodes the normal ASLV-A receptor on chicken cells (1). Taken together, these results demonstrate that the cloned gene encodes a subgroup A virus receptor.Since it is clear that the cloned gene encodes an ASLV-A receptor, the protein supposed to be encoded by the classical TVA locus, we asked whether the identified receptor gene mapped to the TVA locus. In this paper we show that a restriction fragment length polymorphism (RFLP) of DNA from inbred chickens can be used to demonstrate that the cloned ASLV-A receptor gene maps to within 5 centimorgans (cM) of the TVA locus as defined by susceptibility to ASLV-A, making it highly probable that the cloned gene is TVA. In addition, analysis of the genomic sequences encoding the viral interaction domain of the receptor in both inbred and randomly bred chickens demonstrates that alterations in this region of the receptor are not responsible for the resistance phenotype.

TVA segregation analysis.

Inbred chicken lines homozygous for TVA alleles encoding either sensitivity (TVA*S) or resistance (TVA*R) were used to generate birds in which the TVA segregation pattern could be studied. Regional Poultry Research Lab lines 6₃ (TVA*S/*S TVB*S/*S) and 7₂ (TVA*R/*R TVB*R/*R) (6) were crossed, and the resulting heterozygous F₁ progeny were mated to generate 93 F₂ chicks. The susceptibility phenotype of the F₂ chicks was determined on the basis of tumor formation following subcutaneous injection of 500 focus-forming units (FFU) of subgroup A Bryan high-titer Rous sarcoma virus (RSV) (RAV-1) and 500 FFU of subgroup B Bryan high-titer RSV (RAV-2) into the right and left wing webs, respectively, of 4-week-old chicks. At 2, 3, and 4 weeks postinjection the wings were palpated to check for tumor development. Chicks were scored as positive for susceptibility (e.g., TVA*S/*S or TVA*S/*R) if a tumor of any size formed in the appropriate wing web.Table ​Table11 summarizes the distribution of susceptibility to subgroups A and B RSV in the F₂ chicks. Segregation of TVB yielded roughly the predicted frequency of susceptible (67 observed versus 70 expected) and resistant (26 observed versus 23 expected) progeny for a dominant locus (P > 0.05). In contrast, the distribution of subgroup A-susceptible and -resistant birds deviated significantly from the expected 3:1 ratio, with an excessive number of subgroup A-resistant chicks observed and a ratio of 2:1 (χ² = 3.4) (Table ​(Table1).1). Previous analysis of the segregation of TVA using the same chicken lines did not reveal an altered distribution of sensitive and resistant birds (6), suggesting that the bias seen here may be a chance deviation from the expected ratio. Segregation of two endogenous virus loci, ALVE2 (previously designated ev2) (carried by line 7₂) and ALVE3 (previously designated ev3) (carried by line 6₃), also gave roughly the expected 3:1 ratio in these F₂ chicks (data not shown). When the segregation bias in the ASLV-A-susceptible birds was accounted for, then susceptibility to subgroup A and B RSV segregated independently, as was expected since the TVA and TVB genes have been reported to be unlinked (6). Recent mapping studies also confirmed that TVA and TVB are found on different linkage groups (3a).

TABLE 1

Segregation of resistance and susceptibility to subgroup A and B viruses in F₂ progeny obtained by crossing lines 6₃ and 7₂

Messenger Activity of Virion RNA for Avian Leukosis Viral Envelope Glycoprotein

An intracellular assay for viral envelope glycoprotein (env) messenger was employed to analyze the RNA from virus particles of Rous-associated virus type 2. For this assay RNA was microinjected into cells infected by the env-deficient Bryan strain of Rous sarcoma virus [RSV(-) cells]. Only when the injected RNA could be translated by the recipient cells to produce viral envelope glycoprotein was the env deficiency of the RSV(-) cells complemented, enabling them to release focus-forming virus. RNA in a 21S size fraction from the Rous-associated virus particle promoted the release of numerous focus-forming virus from RSV(-) cells, whereas the major 35S virion RNA species was inactive. The env messenger activity sedimented as a sharp peak with high specific activity. RNase T1-generated fragments of virion 35S RNA were unable to promote the release of infectious virus from RSV(-) cells. Consequently, the active molecule was most likely to be env messenger which had been encapsulated by the virus particle from the cytoplasm of infected cells. Approximately 95% of the env messenger within the virion was associated with the virion high-molecular-weight RNA complex. The temperature required to dissociate env messenger from the high-molecular-weight complex was indistinguishable from the temperature required to disrupt the complex itself. Virion high-molecular-weight RNA that was associated with env messenger sedimented slightly more rapidly than the bulk virion RNA; this was the strongest evidence that the 21S messenger had been encapsulated directly from the infected cells. These data are considered along with a related observation [concerning the prolonged expression of env messenger after injection into RSV(-) cells] to raise the possibility that virus-encapsulated env messenger can become expressed within subsequently infected cells.     

Mutational Analysis of the Putative Fusion Domain of Ebola Virus Glycoprotein

Ebola viruses contain a single glycoprotein (GP) spike, which functions as a receptor binding and membrane fusion protein. It contains a highly conserved hydrophobic region (amino acids 524 to 539) located 24 amino acids downstream of the N terminus of the Ebola virus GP2 subunit. Comparison of this region with the structural features of the transmembrane subunit of avian retroviral GPs suggests that the conserved Ebola virus hydrophobic region may, in fact, serve as the fusion peptide. To test this hypothesis directly, we introduced conservative (alanine) and nonconservative (arginine) amino acid substitutions at eight positions in this region of the GP2 molecule. The effects of these mutations were deduced from the ability of the Ebola virus GP to complement the infectivity of a vesicular stomatitis virus (VSV) lacking the receptor-binding G protein. Some mutations, such as Ile-to-Arg substitutions at positions 532 (I532R), F535R, G536A, and P537R, almost completely abolished the ability of the GP to support VSV infectivity without affecting the transport of GP to the cell surface and its incorporation into virions or the production of virus particles. Other mutations, such as G528R, L529A, L529R, I532A, and F535A, reduced the infectivity of the VSV-Ebola virus pseudotypes by at least one-half. These findings, together with previous reports of liposome association with a peptide corresponding to positions 524 to 539 in the GP molecule, offer compelling support for a fusion peptide role for the conserved hydrophobic region in the Ebola virus GP.     

Visualization of the Two-Step Fusion Process of the Retrovirus Avian Sarcoma/Leukosis Virus by Cryo-Electron Tomography

Retrovirus infection starts with the binding of envelope glycoproteins to host cell receptors. Subsequently, conformational changes in the glycoproteins trigger fusion of the viral and cellular membranes. Some retroviruses, such as avian sarcoma/leukosis virus (ASLV), employ a two-step mechanism in which receptor binding precedes low-pH activation and fusion. We used cryo-electron tomography to study virion/receptor/liposome complexes that simulate the interactions of ASLV virions with cells. Binding the soluble receptor at neutral pH resulted in virions capable of binding liposomes tightly enough to alter their curvature. At virion-liposome interfaces, the glycoproteins are ∼3-fold more concentrated than elsewhere in the viral envelope, indicating specific recruitment to these sites. Subtomogram averaging showed that the oblate globular domain in the prehairpin intermediate (presumably the receptor-binding domain) is connected to both the target and the viral membrane by 2.5-nm-long stalks and is partially disordered, compared with its native conformation. Upon lowering the pH, fusion took place. Fusion is a stochastic process that, once initiated, must be rapid, as only final (postfusion) products were observed. These fusion products showed glycoprotein spikes on their surface, with their interiors occupied by patches of dense material but without capsids, implying their disassembly. In addition, some of the products presented a density layer underlying and resolved from the viral membrane, which may represent detachment of the matrix protein to facilitate the fusion process.     

蛋鸡J亚群禽白血病的分子生物学诊断

根据J亚群白血病病毒(ALV-J)原型株HPRS-103的序列设计了一对针对外源性ALV-J引物H5和H7,从发生ML病死鸡的肿瘤、骨髓、肝脏、脾脏和输卵管组织中提取DNA作为模板,经PCR扩增得到长度为545bp的片段,对其序列进行测定后,与ALV-J原型株HPRS-103的序列进行了比较,发现其核苷酸同源性为97．4％,所编码氨基酸的同源性为96．1％。该片段含有ALV-J gp85编码基因的部分序列和ALV-J pol基因的部分序列,从分子水平上证实了蛋鸡发生J亚群禽白血病,进一步证明了此前根据病理学观察、免疫组化及免疫荧光诊断的结果。这是首次从分子水平上证明蛋用型鸡发生J亚群禽白血病。     

Isolation and Identification of a Subgroup A Avian Leukosis Virus from Imported Meat-type Grand-parent Chickens

An exogenous avian leukosis virus (ALV) strain SDAU09C1 was isolated in DF-1 cells from one of 240 imported 1-day-old white meat-type grand parent breeder chicks. Inoculation of SDAU09C1 in ALV-free chickens induced antibody reactions specific to subgroup A or B. But gp85 amino acid sequence comparisons indicated that SDAU09C1 fell into subgroup A; it had homology of 88.8%-90.3% to 6 reference strains of subgroup A, much higher compared to other subgroups including subgroup B. This is the first report for A...     

The Unique Envelope Gene of the Subgroup J Avian Leukosis Virus Derives from ev/J Proviruses, a Novel Family of Avian Endogenous Viruses

A new subgroup of avian leukosis virus (ALV), designated subgroup J, was identified recently. Viruses of this subgroup do not cross-interfere with viruses of the avian A, B, C, D, and E subgroups, are not neutralized by antisera raised against the other virus subgroups, and have a broader host range than the A to E subgroups. Sequence comparisons reveal that while the subgroup J envelope gene includes some regions that are related to those found in env genes of the A to E subgroups, the majority of the subgroup J gene is composed of sequences either that are more similar to those of a member (E51) of the ancient endogenous avian virus (EAV) family of proviruses or that appear unique to subgroup J viruses. These data led to the suggestion that the ALV-J env gene might have arisen by multiple recombination events between one or more endogenous and exogenous viruses. We initiated studies to investigate the origin of the subgroup J envelope gene and in particular to determine the identity of endogenous sequences that may have contributed to its generation. Here we report the identification of a novel family of avian endogenous viruses that include env coding sequences that are over 95% identical to both the gp85 and gp37 coding regions of subgroup J viruses. We call these viruses the ev/J family. We also report the isolation of ev/J-encoded cDNAs, indicating that at least some members of this family are expressed. These data support the hypothesis that the subgroup J envelope gene was acquired by recombination with expressed endogenous sequences and are consistent with acquisition of this gene by only one recombination event.     

Polymorphism of Avian Leukosis Virus Subgroup E Loci Showing Selective Footprints in Chicken

Avian leukosis virus subgroup E (ALVE) is a family of endogenous retroviruses in the chicken genome. To investigate the genetic consequences of chicken domestication, we analyzed 18 ALVE loci in red jungle fowls, layers, broilers, and Chinese indigenous chickens. None of the ALVE loci tested were found in red jungle fowls, but 12 were present in domestic chickens. ALVE1 and ALVE16 are found in regions of the genome that harbor quantitative trait loci (QTL) affecting egg production traits. ALVE1 was fixed and ALVE16 was detected only in layers. By contrast, ALVE-b1, ALVE-b5, ALVE-b6, and ALVE-b8 integrated into regions of the genome that harbor QTL affecting meat production traits. Carrier frequencies of these four ALVE loci were high in broilers and low in Chinese local chickens; the loci were not found in the layers. This study demonstrated that insertionally polymorphic ALVE loci can illustrate the selective footprints in the chicken genome.     

黄羽肉鸡J亚群白血病病毒的分子生物学特性和致病性

通过接种鸡胚成纤维细胞(CEF)、聚合酶链式反应(PCR)技术及特异性单抗的间接荧光抗体反应(IFA),首次从中国地方品系-黄羽肉鸡中分离到J亚群白血病病毒(ALV-J),并对其gp85基因和3′Ter序列及其致病性作了比较分析。结果显示,GD0510A、GD0510B和GD0512的gp85基因编码的氨基酸序列与国内毒株HN0001同源性最高,分别为94.1%、92.5%和95.8%,GD0510A和GD0512的3′Ter核酸序列与国内毒株同源性比较高。分离到的两株ALV-J(GD0510A和GD0512)感染1日龄肉鸡后出现明显生长抑制(P<0.05),并诱发中枢免疫器官法氏囊和胸腺萎缩。两株病毒单独感染均能降低鸡体对新城疫病毒和禽流感病毒(AIV-H5)疫苗的抗体滴度,GD0512感染鸡后能明显抑制感染鸡对新城疫病毒疫苗的免疫反应(P<0.05),而GD0510A感染鸡后在4w时也能明显抑制感染鸡对禽流感病毒(AIV-H5)疫苗的免疫反应(P<0.05)。研究证实在我国地方品系黄羽肉鸡存在ALV-J的感染,分子生物学特性研究表明该毒株可能是来自白羽肉鸡且能造成感染鸡的免疫抑制。     

A Recombinant Avian Leukosis Virus Subgroup J for Directly Monitoring Viral Infection and the Selection of Neutralizing Antibodies

Avian leukosis virus subgroup J (ALV-J) has induced serious clinical outbreaks and has become a serious infectious disease of chickens in China. We describe here the creation of a recombinant ALV-J tagged with the enhanced green fluorescent protein (named rHPRS-103EGFP). We successfully utilize the rHPRS-103EGFP to visualize viral infection and for development of a simplified serum-neutralization test.     

Independent Isolates of the Emerging Subgroup J Avian Leukosis Virus Derive from a Common Ancestor

A new subgroup of avian leukosis virus (ALV) that includes a unique env gene, designated J, was identified recently in England. Sequence analysis of prototype English isolate HPRS-103 revealed several other unique genetic characteristics of this strain and provided information that it arose by recombination between exogenous and endogenous virus sequences. In the past several years, ALV J type viruses (ALV-J) have been isolated from broiler breeder flocks in the United States. We were interested in determining the relationship between the U.S. and English isolates of ALV-J. Based on sequence data from two independently derived U.S. field isolates, we conclude that the U.S. and English isolates of ALV-J derive from a common ancestor and are not the result of independent recombination events.     

Detection and Molecular Characterization of J Subgroup Avian Leukosis Virus in Wild Ducks in China

To assess the status of avian leukosis virus subgroup J (ALV-J) in wild ducks in China, we examined samples from 528 wild ducks, representing 17 species, which were collected in China over the past 3 years. Virus isolation and PCR showed that 7 ALV-J strains were isolated from wild ducks. The env genes and the 3′UTRs from these isolates were cloned and sequenced. The env genes of all 7 wild duck isolates were significantly different from those in the prototype strain HPRS-103, American strains, broiler ALV-J isolates and Chinese local chicken isolates, but showed close homology with those found in some layer chicken ALV-J isolates and belonged to the same group. The 3′UTRs of 7 ALV-J wild ducks isolates showed close homology with the prototype strain HPRS-103 and no obvious deletion was found in the 3′UTR except for a 1 bp deletion in the E element that introduced a binding site for c-Ets-1. Our study demonstrated the presence of ALV-J in wild ducks and investigated the molecular characterization of ALV-J in wild ducks isolates.