A new class of endogenous human retroviral genomes

Human DNA contains multiple copies of a novel class of endogenous retroviral genomes. Analysis of a human recombinant DNA clone (HLM-2) containing one such proviral genome revealed that it is a mosaic of retroviral-related sequences with the organization and length of known endogenous retroviral genomes. The HLM-2 long terminal repeat hybridized with the long terminal repeat of the squirrel monkey virus, a type D retrovirus. The HLM-2 gag and pol genes share extensive nucleotide sequence homology with those of the M432 retrovirus (a type A-related retrovirus), mouse mammary tumor virus (a type B retrovirus), and the avian Rous sarcoma virus (a type C retrovirus). Nucleotide sequence analysis revealed regions in the HLM-2 pol gene that were as much as 70 percent identical to the mouse mammary tumor virus pol gene. A portion of the putative HLM-2 env gene hybridized with the corresponding region of the M432 viral genome.     

禽白血病病毒多重PCR检测法的建立及初步应用

根据GenBank中登录的禽白血病病毒A、B、J亚群的核苷酸序列设计合成了3对引物,各对引物可分别扩增出大小约为195、260、924bp的核苷酸片段;经优化建立禽白血病病毒多重PCR检测法,并进行其敏感性试验及对马立克病病毒(MDV)、火鸡疱疹病毒(HVT)、新城疫病毒(NDV)、传染性囊病病毒(IBDV)、传染性喉气管炎病毒(ILTV)、传染性支气管炎病毒(IBV)和DF-1细胞基因组的特异性试验;应用该方法对1 294份临床样品进行禽白血病病毒检测,对阳性检出样品抽样进行测序鉴定.结果表明:该方法的最小检出拷贝数为103,特异性试验均为阴性,检出临床样品中的核苷酸片段与参考毒株核苷酸序列的同源性达98%以上,符合率100%,具有良好的特异性、敏感性和符合率,可为禽白血病病毒感染的临床诊断及流行病学调查研究提供有效借鉴.     

Avian Leukosis in Brahma Chicken Raised in Al-Qurnah Town,Southern Iraq

The current study was conducted to diagnose avian leukosis in naturally infected Brahma backyard chickens in southern parts of Iraq, on the basis of clincopathological findings and serological detection by using antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) in suspected tumor cases in field conditions. In this study the avian leukosis was mostly observed in birds from 16 to 22 weeks of age, as well as the infected flocks showed a low mortality rate ranging from 5–6%. Typical variable sized grey to yellow obvious tumor-like nodular lesion was demonstrated on the surface of enlarged visceral organs such as liver, spleen, kidney and duodenum, as in white meat-type chickens. The histopathological features revealed massive infiltration of monomorphic lymphocytes in which the lymphoblasts were predominant in the liver, kidney, spleen and duodenum. In this study, a total of 40 sera were tested for ALV P27 antigen by ELISA technique. Thirty-five out of forty sera (87.5%) obtained from Brahma chickens tested positive to ALV P27 antigen and a higher percentage (88.58%) of the chicken sera were strongly positive and had (EUs > 75%). Based on these findings, avian leukosis was concluded to be associated with this pathological condition in Iraqi backyard flocks. This is the first report of the presence of the avian leukosis in visceral samples of Brahma breed. It seems that commercial poultry population in Iraq is not far from the threat of the avian leukosis, and surveillance for avian leukosis is needed.     

New findings on the congenital transmission of avian leukosis viruses

RAV-0, an endogenous avian leukosis virus, does not undergo congenital transmission in infected K28 chickens. In contrast, avian leukosis viruses of exogenous origin undergo highly efficient congenital transmission. The relative abilities of endogenous and exogenous viruses to undergo congenital transmission appear to be determined by the p27 capsid proteins of these viruses.     

PI3K/Akt信号转导通路在ALV-J感染中作用的初步研究

 【目的】探讨ALV-J在宿主细胞中复制与PI3K/Akt信号转导通路的关系。【方法】将血管瘤病变型ALV-J毒株HN06和骨髓瘤病变型ALV-J毒株NX0101分别感染DF-1细胞,通过Western blot、Real-time PCR、IFA和ELISA等方法,观察细胞Akt蛋白磷酸化水平、病毒RNA表达水平和病毒蛋白表达水平等指标。【结果】HN06株和NX0101株在体外细胞中复制水平有差异。HN06株的早期感染可引起Akt转导通路的活化,病毒引起的Akt磷酸化具有病毒滴度依赖性,而且能被PI3K特异性抑制剂LY294002所抑制,表明HN06株诱导的Akt活化是PI3K途径依赖的。LY294002可在病毒感染早期呈剂量依赖性地显著降低受染细胞中HN06 RNA水平、囊膜蛋白水平和细胞培养物上清中的病毒粒子含量。【结论】PI3K/Akt信号转导通路活化对HN06株在细胞感染早期具有重要的作用,该结果与已报道的有关细胞PI3K/Akt信号转导通路参与NX0101株的早期感染的结论一致。本研究为进一步阐明ALV-J入侵宿主细胞和复制的精确机制等研究奠定了基础。     

Efficient incorporation of human CD4 protein into avian leukosis virus particles

Virus envelope (Env) proteins are thought to contain specific signals for selective uptake by virus particles. In the course of attempting to define these signals by testing virus incorporation of CD4-Env chimeric proteins, normal human CD4 was found to be efficiently and selectively assembled into avian leukosis virus particles in quail cells. Viruses bearing CD4 at their surface may be useful reagents in the design of retrovirus-mediated gene therapy for the acquired immune deficiency syndrome.     

Heterogeneity of murine leukemia virus in vitro DNA; detection of viral DNA in mammalian cells

Kinetic analysis of the reassociation of DNA synthesized in vitro by a murine leukemia virus DNA polymerase revealed two classes of double-stranded product representative of 25 and 100 percent of viral genetic information. The DNA product representing the smaller portion of the viral genome comprised 85 percent of the double-stranded DNA generated in vitro and was extensively duplicated in the genomes of both normal cells and cells containing RNA tumor virus.     

Molecular cloning of complementary DNA encoding the avian receptor for vitamin D

Vitamin D3 receptors are intracellular proteins that mediate the nuclear action of the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Two receptor-specific monoclonal antibodies were used to recover the complementary DNA (cDNA) of this regulatory protein from a chicken intestinal lambda gt11 cDNA expression library. The amino acid sequences that were deduced from this cDNA revealed a highly conserved cysteine-rich region that displayed homology with a domain characteristic of other steroid receptors and with the gag-erbA oncogene product of avian erythroblastosis virus. RNA selected via hybridization with this DNA sequence directed the cell-free synthesis of immunoprecipitable vitamin D3 receptor. Northern blot analysis of polyadenylated RNA with these cDNA probes revealed two vitamin D receptor messenger RNAs (mRNAs) of 2.6 and 3.2 kilobases in receptor-containing chicken tissues and a major cross-hybridizing receptor mRNA species of 4.2 kilobases in mouse 3T6 fibroblasts. The 4.2-kilobase species was substantially increased by prior exposure of 3T6 cells to 1,25(OH)2D3. This cDNA represents perhaps the rarest mRNA cloned to date in eukaryotes, as well as the first receptor sequence described for an authentic vitamin.     

鸡J亚型白血病病毒荧光定量PT-PCR检测方法的建立

运用实时荧光定量PCR技术,针对鸡J亚型白血病的gp85基因设计特异性引物,建立了一种快速准确诊断鸡J亚型白血病病毒(ALV-J)的荧光定量PT-PCR方法。通过重复性、特异性和灵敏度等验证表明,该方法适用于ALV-J的临床检测,为ALV-J引起的鸡肿瘤性疾病的快速诊断及采取相应的治疗措施提供了重要的技术支撑。     

Sindbis virus: an efficient, broad host range vector for gene expression in animal cells

Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. The vectors were self-replicating and gene expression was efficient and rapid; up to 10(8) CAT polypeptides were produced per infected cell in 16 to 20 hours. CAT expression could be made temperature-sensitive by means of a derivative that incorporated a temperature-sensitive mutation in viral RNA synthesis. Vector genomic RNAs were packaged into infectious particles when Sindbis helper virus was used to supply virion structural proteins. The vector RNAs were stable to at least seven cycles of infection. The expression of CAT increased about 10(3)-fold, despite a 10(15)-fold dilution during the passaging. Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells.     

实验感染肉鸡组织中J亚群禽白血病病毒的抗原定位

从实验感染J亚群禽白血病病毒(ALVJ)的髓细胞性白血病肉鸡病例中,选取典型病例,用J亚群禽白血病病毒单克隆抗体通过免疫酶组化试验对不同组织进行了抗原定位观察。结果显示:在实验感染阳性肉鸡体内实质组织细胞及瘤细胞的核膜和细胞浆里可见不同程度的棕黄色阳性反应,可为探讨ALVJ对体内组织细胞的嗜性提供依据。     

清远麻祖代鸡群禽白血病感染研究

采用禽白血病抗原检测试剂盒对8、12、20和26周龄的同一清远麻祖代鸡群泄殖腔拭子样品各500份进行ELISA检测,结果显示:抽检鸡群在不同周龄的禽白血病阳性率分别为5.2％、9.4％、12.6％和8.6％,清远麻祖代鸡白血病的排毒高峰在20周龄,是进行禽白血病抗原检测的最佳时期.     

血管瘤型禽白血病的实验室诊断

采用血清学方法和分子生物学技术对某蛋鸡场感染鸡进行实验室诊断,采用禽白血病A或B亚群抗体检测试剂盒对该鸡场的血清样本进行检测,抗体阳性率达到70%(7/10);针对ALV群特异性抗原P27基因进行PCR扩增,血液、肝脏病料样本中核酸的检出率分别为66.7%(4/6)、75%(3/4);取ALV 囊膜糖蛋白gp85 基因的扩增产物进行酶切分析,扩增的目的片断中存在BglⅡ和 SspⅠ酶切位点,BamHⅠ不能切割PCR 产物.试验结果证实本次疫病是由ALV-A 亚群病毒引起的血管瘤型白血病.     

Identification of virus-encoded microRNAs

RNA silencing processes are guided by small RNAs that are derived from double-stranded RNA. To probe for function of RNA silencing during infection of human cells by a DNA virus, we recorded the small RNA profile of cells infected by Epstein-Barr virus (EBV). We show that EBV expresses several microRNA (miRNA) genes. Given that miRNAs function in RNA silencing pathways either by targeting messenger RNAs for degradation or by repressing translation, we identified viral regulators of host and/or viral gene expression.     

DNA of Rous sarcoma virus: its nature and significance

Purified preparations of Rous sarcoma virus (an avian tumor virus with an RNA genome) contain small amounts of double-stranded DNA. This DNA cannot be hybridized to viral RNA, but will reanneal completely with the DNA of avian cells. Extensive substitution of bromodeoxyuridine for thymidine in "viral" DNA does not photosensitize the biological activity of the virus. These observations indicate that the DNA associated with Rous sarcoma virus is derived from the DNA of the avian host cell, and is probably devoid of any function in the life cycle of the virus.     

五华鸡Mx基因A2032G变异位点与禽白血病关联分析

Mx基因是具有抗禽流感病毒作用的基因。为探讨Mx基因A2032G变异位点是否与禽白血病有关,本试验采用PCR-RFLP方法,检测了99只56周龄五华鸡Mx基因多态性,并与禽白血病进行关联分析。检测群体Mx基因2032位点存在2个等位基因A、G,频率分别为3.03%和96.97%;两种基因型AG、GG,频率分别为6.06%和93.94%。卡方适合性检验显示,该群体Mx基因2032位点的分布偏离Hardy-Weinberg平衡。分析这2种基因型与禽白血病阳性率的相关性显示,AG型群体禽白血病阳性率为16.67%低于GG型阳性率58.06%。基于上述结论,则阴性群体中AG型个体多于GG型,以40只1日龄禽白血病阴性鸡群为对照,相同方法检测Mx基因2032位点突变率,共检测到3种基因型AA、AG、GG,其频率分别为2.50%、5.00%和92.50%,与上述结论不符。推测五华鸡GG型为禽白血病易感群体,禽白血病阳性检出率可能随日龄而发生变化,且AG基因型的禽白血病易感率低于GG基因型。     

A cellular microRNA mediates antiviral defense in human cells

In eukaryotes, 21- to 24-nucleotide-long RNAs engage in sequence-specific interactions that inhibit gene expression by RNA silencing. This process has regulatory roles involving microRNAs and, in plants and insects, it also forms the basis of a defense mechanism directed by small interfering RNAs that derive from replicative or integrated viral genomes. We show that a cellular microRNA effectively restricts the accumulation of the retrovirus primate foamy virus type 1 (PFV-1) in human cells. PFV-1 also encodes a protein, Tas, that suppresses microRNA-directed functions in mammalian cells and displays cross-kingdom antisilencing activities. Therefore, through fortuitous recognition of foreign nucleic acids, cellular microRNAs have direct antiviral effects in addition to their regulatory functions.     

一株J亚型禽白血病病毒的分离鉴定及其gp85基因的序列分析

为确定安徽巢湖某地方品种养鸡场的疑似禽白血病(avian leukosis,AL)的病原,以DF-1细胞从肿瘤组织中分离病毒,采用RT-PCR方法进行鉴定,并对其感染细胞上清进行透射电镜观察。同时对送检病鸡的心、肝、脾及腺胃等进行组织病理学观察,最后将扩增出的gp85基因与GenBank收录的其他ALV序列进行比对分析。结果表明:PCR扩增产物大小约为928 bp,经测序证实为ALV gp85基因,将该分离株命名为AHCH-2018株。透射电镜观察到具有囊膜的大小约100 nm的病毒粒子。组织病理学结果显示,病鸡的心、肝、脾及腺胃中有大量成灶淋巴细胞增殖。遗传进化分析结果表明,分离株的gp85基因与多株ALV J参考毒株核苷酸序列的同源性为94.1%~99.5%,且与GD1401J株的核苷酸同源性最高,达99.5%,与A、B、C、D、E亚群同源性为46.5%~49.7%。该分离株与J亚群原型毒株处于同一大分支。本研究为了解安徽土鸡种群中禽白血病的分子流行病学特点提供了一定的数据参考。