Expression of Endogenous Retrovirus ev/J gp85 Gene and Analysis of Its Immunoreactivity in Comparison with Exogenous Viral Protein

The envelope gene gp85 of ev/J,a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian ieukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC<,10200> strain).     

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     

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.     

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。     

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.     

Segregation of EAV-HP Ancient Endogenous Retroviruses within the Chicken Population

Avian leukosis virus subgroup J (ALV-J), an exogenous avian retrovirus, is thought to have evolved by recombination with the highly identical env gene of the endogenous avian retrovirus EAV-HP. Embryonic expression of EAV-HP env has been suggested to be associated with the induction of immunological tolerance, a feature observed in a significant proportion of meat-type chickens infected with ALV-J. In support of this hypothesis, we demonstrate that EAV-HP loci, some of which could be associated with tolerance, are still segregating within the chicken population.     

In vivo analysis of <Emphasis Type="Italic">Drosophila</Emphasis> SU(<Emphasis Type="Italic">Z</Emphasis>)12 function

Polycomb group (PcG) proteins are required to maintain a stable repression of the homeotic genes during Drosophila development. Mutants in the PcG gene Supressor of zeste 12 (Su(z)12) exhibit strong homeotic transformations caused by widespread misexpression of several homeotic genes in embryos and larvae. Su(z)12 has also been suggested to be involved in position effect variegation and in regulation of the white gene expression in combination with zeste. To elucidate whether SU(Z)12 has any such direct functions we investigated the binding pattern to polytene chromosomes and compared the localization to other proteins. We found that SU(Z)12 binds to about 90 specific eukaryotic sites, however, not the white locus. We also find staining at the chromocenter and the nucleolus. The binding along chromosome arms is mostly in interbands and these sites correlate precisely with those of Enhancer-of-zeste and other components of the PRC2 silencing complex. This implies that SU(Z)12 mainly exists in complex with PRC2. Comparisons with other PcG protein-binding patterns reveal extensive overlap. However, SU(Z)12 binding sites and histone 3 trimethylated lysine 27 residues (3meK27 H3) do not correlate that well. Still, we show that Su(z)12 is essential for tri-methylation of the lysine 27 residue of histone H3 in vivo, and that overexpression of SU(Z)12 in somatic clones results in higher levels of histone methylation, indicating that SU(Z)12 is rate limiting for the enzymatic activity of PRC2. In addition, we analyzed the binding pattern of Heterochromatin Protein 1 (HP1) and found that SU(Z)12 and HP1 do not co-localize.     

Genome structure and expression of the ev/J family of avian endogenous viruses.

We recently reported the identification of sequences in the chicken genome that show over 95% identity to the novel envelope gene of the subgroup J avian leukosis virus (S. J. Benson, B. L. Ruis, A. M. Fadly, and K. F. Conklin, J. Virol. 72:10157-10164, 1998). Based on the fact that the endogenous subgroup J-related env genes were associated with long terminal repeats (LTRs), we concluded that these LTR-env sequences defined a new family of avian endogenous viruses that we designated the ev/J family. In this report, we have further characterized the content and expression of the ev/J proviruses. The data obtained indicate that there are between 6 and 11 copies of ev/J proviruses in all chicken cells examined and that these proviruses fall into six classes. Of the 18 proviruses examined, all share a high degree of sequence identity and all contain an internal deletion that removes all of the pol gene and various amounts of gag and env gene sequences. Sequencing of the gag genes, LTRs, and untranslated regions of several ev/J proviruses revealed a high level of identity between isolates, indicating that they have not undergone significant sequence variation since their introduction into the avian germ line. Although the ev/J gag gene showed a relatively weak relationship (46% identity and 61% similarity at the amino acid level) to that of the avian leukosis-sarcoma virus family, it retains several sequences of demonstrated importance for virus assembly, budding, and/or infectivity. Finally, evidence was obtained that at least some members of the ev/J family are expressed and, if translated, could encode Gag- and Env-related polypeptides.     

Transposable elements behavior following viral genomic stress inDrosophila melanogaster inbred line

To analyze the behavior of endogenous transposable elements under genomic stress, aDrosophila melanogaster inbred line was submitted to three kinds of viral perturbations. First, a retroviral plasmid containing the avian Rous Associated Virus type 2 (RAV-2) previously deleted for the viral envelope coding gene (env) was introduced by P element transformation into theDrosophila genome. An insertion of this avian retroviral sequence was detected byin situ hybridization in site 53C on polytene chromosome arm 2R. Second,Drosophila embryos were injected with RAV-2 particles produced by cell culture after transfection with the retroviral plasmid. Third, theDrosophila melanogaster inbred line was stably infected by the sigma native virus. It appears that neither the offspring of the flies in which the viral DNA was found integrated nor those from the infected sigma flies showed copia or mdgl element mobilization. Injection of the avian RAV-2 particles led, however, to the observation of somatic transpositions of mdgl element on the 2L chromosome, the copia element insertion pattern remaining stable. Thus, endogenous transposable elements show more instability in sublines injected with exogenous viral particles than in a transgenic subline containing a foreign viral insert, all transposable elements not being equally sensitive to such genomic stress. Correspondence to: I. Jouan-Dufournel     

Sequence variation in the gp120 region of SHIV-CN97001 during in vivo passage

SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified by RT-PCR from the plasma of SHIV-CN97001 infected animals at the peak viral load time point and the gene distances (divergence, diversity) were calculated using DISTANCE. The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest during in vivo passage. It had a relationship between viral divergence from the founder strain and viral replication ability. The nucleic acid sequence of the V3 region was highly conservative. All of the SHIV-CN97001 strains had V3 loop central motif (GPGQ) and were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop. These results show that there was no significant increase in the genetic distance during serial passage, and SHIV-CN97001 gp120 gene evolved toward ancestral states upon transmission to a new host. This could partly explain why there was no pathogenic viral strain obtained during in vivo passage. Foundation items: CIPRA (U19 AI051915); 973 (2005CB-522903).     

Different quasispecies with great mutations hide in the same subgroup J field strain of avian leukosis virus

Blood samples were collected from a local strain of chickens associated with serious tumor cases in Shandong Province.The samples were inoculated into chicken embryo fibroblast and DF-1 cells for virus isolation and identification,respectively.The inoculated cells were screened for three common chicken tumor viruses.Nine strains of avian leukosis virus subgroup J(ALV-J) were identified,and were designated LY1201‐LY1209.The env gene from the LY1201 strain was amplified and cloned.All nine resultant env clones(clones 01-09) were sequenced,and the gp85 and gp37 amino acid regions were subjected to homology analysis.Clones 01 and 03 had 10 amino acid deletions in the gp85 region compared to the other seven clones,suggesting that at least two quasispecies with obvious mutations coexist in the same field strain.Among these nine clones,three had identical gp85 and gp37 sequences,and were recognized as the dominant LY1201 quasispecies.The amino acid sequence homology of gp37 and gp85 among the nine clones was 98.5%-100.0% and 96.6%-100.0% respectively,suggesting that the gp85 region of the env gene can better display the quasispecies diversity of ALV-J than gp37.     

The monoclonal antibody JIM19 modulates abscisic acid action in barley aleurone protoplasts

A panel of hybridoma products generated against pea (Pisum sativum L.) guard-cell protoplasts has been assayed for anti-abscisic acid (ABA) biological activity in barley (Hordeum vulgare L.) aleurone protoplasts. The effects of the antibodies on ABA-induced accumulation of mRNA transcribed from RAB-16, a gene responsive to ABA, were determined. Most of the antibodies, and culture medium, had no effect, but five monoclonal antibodies (MAbs) were found to inhibit ABA-induced RAB-16 gene expression and one MAb enhanced it. The effects of one inhibitory MAb, JIM19, were studied in some detail. These effects were specific to ABA-induced events, as incubation with JIM19 had no effect on the expression of a constitutively-expressed gene, GAPDH, encoding glyceraldehyde-3-phosphate dehydrogenase, and only a slight effect on the production of -amylase induced by gibberellic acid. Increasing concentrations of ABA in the incubation medium partly overcame the inhibitory effect of JIM19. Immunolabelling and biological activity remained together during immuno-purification of JIM19 from hybridoma culture supernatant. Immunoblotting of JIM19 to membrane preparations from barley aleurone protoplasts revealed that JIM19 recognised a number of proteins.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid - GAPDH gene encoding glyceraldehyde-3-phosphate dehydrogenase - GCP guard-cell protoplast - MAb monoclonal antibody - RAB (gene) responsive to ABA We thank the Agricultural and Food Research Council and The Nuffield Foundation for financial support, Professor Keith Roberts (John Innes Institute, Norwich, UK) for advice and generous use of his laboratory and Jan Peart (John Innes Institute) for animal cell culture. S.J.N. is grateful to Professor Colin Hawkes (University of the West of England, Bristol) for his continued support of this project.     

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying theNeo ^r selectable marker and theEscherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G₁ progeny at a frequency of 2.7%.Neo ^r andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G₂ progeny.     

Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with<Emphasis Type="Italic">HMG I/Y</Emphasis> as an endogenous reference gene

In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T₀) were calculated by comparing threshold cycle (C _T) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsC _T value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.     

Avian leukosis virus subgroup J infection influences the gut microbiota composition in Huiyang bearded chickens

Avian leukosis virus (ALV) poses a major threat to poultry. The chicken gut microbiota plays critical roles in host performance, health and immunity. However, the effect of viral infection on the microbiota of Chinese local chickens is not well understood. In this study, we performed high-throughput 16S rRNA gene sequencing and evaluated the gut microbiota profiles using faeces from ALV subgroup J (ALV-J)-infected and healthy Huiyang bearded chickens (Chinese local chickens). At the phylum level, ALV-J infection mainly increased the abundance of Bacteroidetes and Proteobacteria and decreased that of Firmicutes. An analysis at the order, family and genus levels showed that the abundance of Lactobacillales, Lactobacillaceae and Lactobacillus was the highest in normal chicken faeces, accounting for 89·07%, 86·47% and 86·46%, respectively, of phylotypes. Moreover, samples from ALV-J-infected chickens were enriched with Bacteroidales, Clostridiales, Bacteroidaceae, Ruminococcaceae, Lachnospiraceae and Bacteroides. Our findings highlight that ALV-J infection alters the gut microbiota and disrupts the host–microbial homeostasis in chickens, which may be involved in the pathogenesis of ALV-J infection.     

A common allergenic epitope of Bermuda grass pollen shared by other grass pollens

The present study disclosed the cross-reactivity between Bermuda grass pollen (BGP) and other grass pollens using monoclonal antibodies (MAbs) and polyclonal antiserum. MAb 9–13, directed against a group of minor allergens of BGP (Cyn d Bd68K, 48K, 38K) was found to cross-react with extracts of ten other grass pollens. Immunoblotting assays illustrated that MAb 9–13 cross-reacted with multiple components of most of these pollens, and the major cross-reactive components had molecular weights of 29–36 kD. The cross-reactivity between BGP andLol pI, the group I allergen of rye grass pollen, was further evaluated;Lol pI was recognized by MAb 9–13, but not by our MAbs/polyclonal antiserum againstCyn dI, the major allergen of BGP. These results suggest that the epitope recognized by MAb 9–13 is a common (C) epitope shared byLol pI andCyn d Bd68K, 48K, 38K, andCyn dI does not share significant antigenicity withLol pI. In a modified radio-allergosorbent test, IgE antibodies in the serum of BGP-allergic patients reacted mildly with C-epitope-bearing components of both BGP and rye grass pollens, and this binding could be blocked specifically by MAb 9–13. This suggests that in addition to an antigenic cross-reaction, the C epitope can also lead to an allergenic cross-reaction.     

斜纹夜蛾核多角体病毒gp37及其邻近序列的克隆及分析

从斜纹夜蛾核多角体病毒(Spodoptera litura nucleopolyhedrovirus, SpltMNPV)基因组中克隆了gp37基因.分子生物学软件分析表明,在gp37基因内部存在糖基化位点.含有晚期表达基因的启动子序列(ATAAG),推测为晚期表达的病毒膜糖蛋白基因.通过GP37蛋白的同源性比较,绘制了以gp37基因为基础的杆状病毒进化树, 发现与以多角体蛋白基因（polyhedrin）为基础所绘制的杆状病毒分子进化树有较大差异.以polyhedrin基因为基础绘制的杆状病毒分子进化树将家蚕核多角体病毒（Bombyx mori nucleopolyhedrovirus, BmNPV）与杆状病毒代表种——苜蓿丫纹夜蛾核多角体病毒（Autographa californica multinucleocapsid nucleopolyhedrovirus, AcMNPV）分开,根据gp37基因分析则将二者归到同一分枝,这与以egt基因为基础绘制的杆状病毒分子进化树结果一致.