Molecular Characterization of the Duck Enteritis Virus UL4 Gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.     

鸭肠炎病毒UL6基因的分子特征

为了研究鸭肠炎病毒(Duck enteritis virus,DEV)UL6的特征,以DEV Clone-03株基因组DNA为模板,用靶基因步移PCR方法扩增得到UL6基因区域2534 bp的DNA片段。结果发现,DEV Clone-03 UL6基因由2373个核苷酸组成,编码一条790个氨基酸残基组成的多肽。与14株已报道的具有全UL6同源基因序列的疱疹病毒参考株进行比较,DEV Clone-03株与α疱疹病毒的核苷酸和氨基酸同源性较之与β疱疹病毒和γ疱疹病毒要高。氨基酸序列比较显示,DEV Clone-03 UL6基因具有α疱疹病毒UL6同源基因5个保守区域。UL6基因推导的氨基酸系统发育进化树分析发现,DEV Clone-03与α疱疹病毒属一个群,在α疱疹病毒中与马立克氏病病毒(Marek′sdisease herpesvirus,MDV)更接近。同时,序列分析发现,在201~222和425~441等氨基酸位,α疱疹病毒参考毒株均有不同程度的缺失,而DEV Clone-03表现出独有的完整性。     

Molecular cloning and characterization of the UL31 gene from Duck enteritis virus

Using a combination of bioinformation analysis and Dot blot technique, a gene, designated hereafter as the duck enteritis virus (DEV) UL31 gene (GenBank accession number EF643559), was identified from the DEV CHv genomic library. Then, the UL31 gene was cloned and sequenced, which was composed of 933 nucleotides encoding 310 amino acids. Multiple sequence alignment suggested that the UL31 gene was highly conserved in Alphaherpesvirinae and similar to the other herpesviral UL31. Phylogenetic analysis showed that the gene had a close evolutionary relationship with the avian herperviruses, and DEV should be placed into a single cluster within the subfamily Alphaherpesvirinae. Antigen prediction indicated that several potential B-cell epitopes sites located in the UL31 protein. To further study, the UL31 gene was cloned into a pET prokaryotic expression vector and transformed into Escherichia coli BL21 (DE3). A 55 kDa fusion protein was induced by the further culture at 37°C after addition of 0.8 mM IPTG. Polyclonal antibody raised against the recombinant UL31 from rabbit was prepared. A protein about 55 kDa that reacted with the antibody was detected in immunoblots of bacterial proteins, suggesting that the 55 kDa protein was the product of the UL31 gene. Immunofluorescence analysis revealed that the protein was localized in very fine punctate forms in the nuclei of infected cells. Our results may provide some insight for further research about the gene and also enrich the database of herpesvirus.     

几种PCR方法在克隆鸭肠炎病毒未知基因中的应用

以DEV基因组DNA为模板, 用简并PCR、改良Targeted gene walking PCR、改良的热不对称交错PCR和Long-PCR, 获得了5350 bp、11083 bp和2905 bp三段DEV未知基因片段, DNA序列分析发现包含9个开放阅读框, 将这些序列提交GenBank分别获得的登录号为: EF554396~EF554403。结果表明, 多种PCR方法联合使用可以高效的实现对鸭肠炎病毒未知基因的克隆。     

Dual infection and superinfection inhibition of epithelial skin cells by two alphaherpesviruses co-occur in the natural host

Hosts can be infected with multiple herpesviruses, known as superinfection; however, superinfection of cells is rare due to the phenomenon known as superinfection inhibition. It is believed that dual infection of cells occurs in nature, based on studies examining genetic exchange between homologous alphaherpesviruses in the host, but to date, this has not been directly shown in a natural model. In this report, gallid herpesvirus 2 (GaHV-2), better known as Marek's disease virus (MDV), was used in its natural host, the chicken, to determine whether two homologous alphaherpesviruses can infect the same cells in vivo. MDV shares close similarities with the human alphaherpesvirus, varicella zoster virus (VZV), with respect to replication in the skin and exit from the host. Recombinant MDVs were generated that express either the enhanced GFP (eGFP) or monomeric RFP (mRFP) fused to the UL47 (VP13/14) herpesvirus tegument protein. These viruses exhibited no alteration in pathogenic potential and expressed abundant UL47-eGFP or -mRFP in feather follicle epithelial cells in vivo. Using laser scanning confocal microscopy, it was evident that these two similar, but distinguishable, viruses were able to replicate within the same cells of their natural host. Evidence of superinfection inhibition was also observed. These results have important implications for two reasons. First, these results show that during natural infection, both dual infection of cells and superinfection inhibition can co-occur at the cellular level. Secondly, vaccination against MDV with homologous alphaherpesvirus like attenuated GaHV-2, or non-oncogenic GaHV-3 or meleagrid herpesvirus (MeHV-1) has driven the virus to greater virulence and these results implicate the potential for genetic exchange between homologous avian alphaherpesviruses that could drive increased virulence. Because the live attenuated varicella vaccine is currently being administered to children, who in turn could be superinfected by wild-type VZV, this could potentiate recombination events of VZV as well.     

Characterization of the highly variable <Emphasis Type="Italic">cry</Emphasis> gene regions of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strain ly4a3 by PCR-SSCP profiling and sequencing

Characterization of cry gene contents can help to predict the insecticidal activities of Bacillus thuringiensis isolates and in the searching of new cry genes. PCR-Single-strand conformation polymorphism (SSCP) profiling and sequencing of the highly variable cry gene regions were used to characterize cry gene content of B. thuringiensis strain ly4a3. The highly variable regions with about 1100 bp in sizes were amplified using a degenerate primer pair for cry genes, OL2(d) and OL5(r). A library of the PCR product was constructed, and all white colonies were subjected to PCR using another degenerate primer pair for cry genes, OL3(d) and OL5(r), with products about 250 bp in sizes. Two different profiles were observed based on SSCP profiling for the PCR products. The cry genes in the two corresponding colonies were sequenced and their deduced amino acids showed high identities to Cry1Ab (84.5%∼98.4%) and Cry1I (88.78%∼98.4%), respectively. This method allows the quick characterization of cry gene content of B. thuringiensis isolates and the detection of new cry genes.     

The Oncogenic MicroRNA OncomiR-21 Overexpressed during Marek's Disease Lymphomagenesis Is Transactivated by the Viral Oncoprotein Meq

Gallid herpesvirus 2 (GaHV-2) is an oncogenic herpesvirus that causes T lymphoma in chicken. GaHV-2 encodes a basic leucine zipper (bZIP) protein of the AP-1 family, Meq. Upon formation of homo- or heterodimers with c-Jun, Meq may modulate the expression of viral and cellular genes involved in lymphomagenesis. GaHV-2 also encodes viral microRNAs (miRNAs) involved in latency and apoptosis escape. However, little is known about cellular miRNA deregulation during the development of GaHV-2-associated lymphoma. We determined the cellular miRNA expression profiles of chickens infected with a very virulent strain (RB-1B) or a vaccine strain (CVI988) or noninfected. Among the most deregulated cellular miRNAs, we focused our efforts on gga-miR-21, which is upregulated during GaHV-2 infection. We mapped the gga-miR-21 promoter to the 10th intron of the TMEM49 gene and found it to be driven by AP-1- and Ets-responsive elements. We show here that the viral oncoprotein Meq binds to this promoter, thereby transactivating gga-miR-21 expression. We confirmed that this miRNA targets chicken programmed death cell 4 (PDCD4) and promotes tumor cell growth and apoptosis escape. Finally, gga-miR-21 was overexpressed only during infection with a very virulent strain (RB-1B) and not during infection with a nononcogenic strain (CVI988), providing further evidence for its role in GaHV-2 lymphomagenesis. Our data therefore suggest an additional role for Meq in GaHV-2-mediated lymphomagenesis through the induction of miR-21 expression.     

Molecular cloning and characterization of a profilin gene <Emphasis Type="Italic">BnPFN</Emphasis> from <Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">nigra</Emphasis> that expressing in a pollen-specific manner

Brassica nigra is a newly found invasive species in Zhejiang Province, China. It distributes alongside the roads, in vegetable fields and on riversides. When it blooms, some natives there will suffer from allergic rhinitis. We designed gene-specific primer pairs according to reported profilin genes and successfully isolated their homolog from flower bud cDNA of B. nigra. The gene, designated BnPFN, was submitted to GenBank under accession number EU004073. BnPFN was 405 bp in length encoding 134 amino acids. Expression analysis of BnPFN gene was carried out by means of RT-PCR. The results showed that BnPFN express only in anthers and pollens, and there was no detection in roots, leaves, stems, sepals, petals and pistils. We suggest that BnPFN is a pollen-specific gene and may be responsible for pollen anaphylactic reactions in those invading areas when B. nigra blooms.     

Gene Arrangement within the Unique Long Genome Region of Infectious Laryngotracheitis Virus Is Distinct from That of Other Alphaherpesviruses

The genome of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) comprises ca. 155 kbp of which ca. one-third have been sequenced so far. To gain additional sequence information we analyzed two stretches of 15.5 and 1.9 kbp of the ILTV unique long (U_L) genome region. The larger fragment contains homologs of the herpes simplex virus (HSV) UL23 (thymidine kinase) and UL22 (glycoprotein H) genes followed by five open reading frames (ORF) encoding putative proteins of 334 to 410 amino acids which exhibit no homology to any known herpesvirus protein. RNA analyses showed that these unique ILTV genes are indeed expressed. An origin of replication separates this cluster of unique genes from a conserved gene cluster consisting of the UL45, UL46, UL48, UL49, UL49.5, and UL50 homologs. The absence of UL47 from this position coincides with the localization of a UL47-homologous ORF within the unique short (U_S) region of the ILTV genome (M. Wild, S. Cook, and M. Cochran, Virus Genes 12:107–116, 1996). Within the second analyzed region the ILTV UL21 homolog was found adjacent to the UL44 gene. We thus identified five novel herpesvirus genes in ILTV and present evidence for a large internal inversion in the ILTV U_L region, in contrast to the collinear genomes of other alphaherpesviruses. Interestingly, a similar inversion is also present in the porcine alphaherpesvirus pseudorabies virus.     

A newly constructed primer pair for the PCR amplification,cloningand sequencing of the flagellin (<Emphasis Type="Italic">flaA</Emphasis>) gene from isolatesof urease-negative <Emphasis Type="Italic">Campylobacter lari</Emphasis>

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564–572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70–75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.     

Characterization of the porcine differentially expressed <Emphasis Type="Italic">PDK4</Emphasis> gene and association with meat quality

To investigate the differential expression of genes in the skeletal muscle between Yorkshire and Chinese indigenous breed Meishan pigs, suppression subtractive hybridization was carried out and many genes were proved to be expressed significantly different in the two breeds. One gene highly expressed in Meishan but lowly expressed in Yorkshire specific library, shared strong homology with human pyruvate dehydrogenase kinase 4 (PDK4). Using semi-quantity and quantity PCR, We confirmed its differential expression between the two breeds. Temporal and spatial expression analysis indicated that porcine PDK4 gene is highly expressed in skeletal muscle and the highest in neonatal pigs. Complete cDNA cloning and sequence analysis revealed that porcine PDK4 gene contains an open reading frame of 1,221 bp. The deduced amino acid sequence showed conservation in evolution. A G/A mutation in intron 9 was identified and association analysis showed that it was significantly associated with intramuscular fat, muscle water content.     

Cloning and sequence diversity analysis of <Emphasis Type="Italic">GmHs1</Emphasis><Superscript><Emphasis Type="Italic">pro-1</Emphasis></Superscript> in Chinese domesticated and wild soybeans

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is the most important pathogen in soybean production worldwide and causes substantial yield losses. An apparent narrow genetic base of SCN resistance was observed in current elite soybean cultivars, and searching for novel SCN resistance genes as well as novel resistance sources rather than focusing on the two important genes rhg1 and Rhg4 has become another major objective in soybean research. In the present paper we report a 1,477 bp Hs1 ^pro-1 homolog, named GmHs1 ^pro-1 . This gene was cloned from soybean variety Wenfeng 7 based on information for Hs1 ^pro-1 , a beet cyst nematode resistance gene in sugar beet. It has two domains, Hs1pro-1_N and Hs1pro-1_C, both of which are believed to confer resistance to nematodes. Of the 1,477 bp sequence in GmHs1 ^pro-1 , an open reading frame of 1,314 bp, encoding a protein with 437 amino acids, was flanked by a 5′-untranslated region of 27 bp and a 3′-untranslated region of 135 bp. Fourteen single-nucleotide polymorphisms (SNPs) were observed in 44 soybean accessions including 23 wild soybeans, 8 landraces, and 13 soybean varieties (or lines), among which 5 in wild soybeans and 3 in landrace accessions were unique. Sequence diversity analysis on the 44 soybean accessions showed π = 0.00168 and θ = 0.00218 for GmHs1 ^pro-1 ; landraces had the highest diversity, followed by wild soybeans, with varieties (or lines) having the lowest. Although we did not detect a significant effect of selection on GmHs1 ^pro-1 in the three populations, sequence diversity, unique SNPs, and phylogenetic analysis indicated a slight domestication bottleneck and an intensive selection bottleneck. High sequence diversity, more unique SNPs, and broader representation across the phylogenetic tree in wild soybeans and landraces indicated that wild collections and landrace accessions are invaluable germplasm for broadening the genetic base of elite soybean varieties resistant to SCN. C. Yuan and G. Zhou contributed to this paper equally.     

伪狂犬病病毒Ea株基因组UL区的克隆与序列分析

从伪狂犬病病毒Ea株基因组DNA中扩增到3．76kb的基因组片段,该片段包含UL31、UL32、UL33和UL34基因完整编码区,以及UL30和UL35基因部分序列。UL31、UL32、UL33和UL34基因G C含量为69．5％～73．4％,偏向于使用富含GC特别是第三密码子位置上核苷酸是C或G的密码子,Ala、Leu、Arg的利用率最高,占氨基酸残基总数的36．4％。PRV Ea株UL31和UL32基因与PRV Ka株核苷酸与氨基酸序列同源性都很高,在98％以上;而UL33和UL34基因与Ka株的氨基酸序列同源性较低,分别为95．7％和94．8％。UL31基因在疱疹病毒α—亚科所有成员之间都很保守,并且UL31基因与马疱疹病毒IV型同源程度最高。UL32、UL33和UL34基因均与牛疱疹病毒I型同源程度最高。UL31、UL32、UL33与UL34基因产物均有酪蛋白激酶2磷酸化位点和蛋白激酶C磷酸化位点,表明UL31、UL32、UL33、UL34蛋白质可能都是磷酸化蛋白质。     

Cloning,Expression and Characterization of the Human <Emphasis Type="BoldItalic">NOB1</Emphasis> gene

The yeast Nob1p (Nin one binding protein) gene is required for proteasome function and RNA metabolism. We report here the cloning and characterization of the human orthologue NOB1 gene and its products. The human NOB1 gene is composed of nine exons and eight introns and is localized on human chromosome 16q22.1. The NOB1 cDNA is 1749 bp long and contains a putative open reading frame of 1239 bp. The predicted NOB1 protein comprises a PIN (PilT amino terminus) domain and a zinc ribbon domain. Western blot analysis showed that the molecular weight of NOB1 is about 50 KDa. RT-PCR analysis of mRNA from human adult tissues showed that NOB1 is expressed mainly in liver, lung and spleen. Expression of NOB1 in mammalian culture cells indicated that the NOB1 protein is mainly localized in the nucleus. Our data provides important information for further study of the function of the NOB1 gene and its products.     

Inhibitory Effect of Resveratrol against Duck Enteritis Virus In Vitro

Duck viral enteritis (DVE) is an acute, contagious herpesvirus infection of ducks, geese, and swans of all ages and species. This disease has been responsible for significant economic losses in domestic and wild waterfowl as a result of mortality, and decreased egg production. Resveratrol is a naturally occurring phytoalexin in specific plants and exhibits inhibitory activity against many kinds of virus. In this paper, resveratrol was found to inhibit duck enteritis virus (DEV) replication in a dose-dependent manner, with a 50% inhibition concentration of 3.85 μg/mL. The inhibition in virus multiplication in the presence of resveratrol was not attributed to direct inactivation or inhibition of virus attachment to the host cells, but to the inhibition of viral multiplication in host cells. The assay of the time of addition limited the drug effect during the first 8 h of infection. This conclusion was supported by the ultrastructure images of the early stage of DEV infection, which showed that the replication of virus nucleic acid and the formation of the capsid in the cell nucleus were suppressed. In the indirect immunofluorescence assay, proteins expression in DEV infected duck embryo fibroblasts (DEFs) within 24 h post-infection (p.i.) was also effectively suppressed by resveratrol. In summary, the resveratrol has a good activity against DEV infection in vitro, which could be attributed to that fact that several essential immediate early viral proteins for virus replication were impacted by resveratrol.     

Identification and characterization of the pseudorabies virus UL3.5 protein, which is involved in virus egress.

Alphaherpesvirus genomes exhibit a generally collinear gene arrangement, and most of their genes are conserved among the different members of the subfamily. Among the exceptions is the UL3.5 gene of pseudorabies virus (PrV) for which positional homologs have been detected in the genomes of varicella-zoster virus, equine herpesvirus 1, and bovine herpesvirus 1 but not in the genomes of herpes simplex virus types 1 and 2. To identify and characterize the predicted 224 amino acid UL3.5 protein of PrV, a rabbit antiserum was prepared against a UL3.5 fusion protein expressed in Escherichia coli. In Western blot (immunoblot) analyses the antiserum detected a 30-kDa protein in the cytoplasm of PrV infected cells which was absent from purified virions. For functional analysis, UL3.5-expressing cell lines were established and virus mutants were isolated after the rescue of defective, glycoprotein B-negative PrV by insertion of the complementing glycoprotein B-encoding gene of bovine herpesvirus 1 at two sites within the UL3.5 locus. A PrV mutant carrying the insertion at codon 159 and expressing a truncated UL3.5 protein was still capable of efficient productive replication in noncomplementing cells. In contrast, a PrV mutant carrying the insertion at codon 10 of the UL3.5 gene did not express detectable UL3.5 protein and exhibited a dramatic growth deficiency on non-complementing cells with regard to plaque formation and one-step replication. Electron microscopical studies showed an accumulation of unenveloped capsids in the vicinity of the Golgi apparatus. This defect could be compensated by propagation on complementing UL3.5-expressing cell lines. Our results thus demonstrate that the PrV UL3.5 gene encodes a nonstructural protein which plays an important role in virus replication, presumably during virus egress. The functionally relevant domains appear to be located within the N-terminal part of the UL3.5 protein which also comprises the region exhibiting the highest level of homology between the predicted UL3.5 homologous proteins of other alphaherpesviruses.