神经坏死病毒在赤点石斑鱼组织中的分布

研究采用地高辛原位杂交和免疫荧光检测技术分别检测病毒RNA2和衣壳蛋白在患病赤点石斑鱼Epinephelus akaara稚鱼中分布。地高辛检测结果表明病毒RNA2主要分布在脑、脊髓、视网膜和鳃上; 免疫荧光检测结果和地高辛检测结果一致, 表明病毒靶器官主要也是脑、脊髓、视网膜和鳃。肠道中几乎检测不到阳性信号, 可能不是病毒的靶器官。因此可以推测神经坏死病毒感染赤点石斑鱼的主要途径是鳃, 而不是肠道。       

PCR扩增同源重组片段筛选转基因胚胎

使用小鼠乳清酸蛋白基因（ＷＡＰ）启动子控制下的人集落刺激因子（Ｇ－ＣＳＦ）基因为显微注射片段,采用ＰＣＲ方法检测了转基因胚,为消除ＰＣＲ扩增中的假阳性结果,构建了两个具有部分同源性的亚克隆片段进行共注射．ＰＣＲ扩增片段跨越这一同源区域,仅当注射的片段能够整合并发生正常重组,转基因整合胚才能以相对高的比例扩增出特异性片段．结果表明,１、２和８细胞期的阳性率分别为１１．１％、５５．５％和４４．４％,较常规ＰＣＲ检测获得更为明确的结论,为在大动物转基因胚胎检测提供了依据     

Sequence of events in natural infection of Pekin duck embryos with duck hepatitis B virus.

The major mode of natural infection of duck hepatitis B virus (DHBV) in Pekin ducks is vertical transmission, with 95 to 100% of the embryos from DHBV-infected dams eventually becoming infected. Maternally transmitted virus is present in large quantities in the yolk of unincubated eggs and is taken up by the embryo during early development. Synthesis of DHBV DNA in the embryo begins at about 6 days of incubation and coincides with the formation of the liver. DHBV DNA synthesis is incomplete, however, until 8 to 10 days of incubation, as shown by comparing the electrophoretic patterns of DHBV-specific nucleic acid species from embryonic livers at successive stages of development. From 8 days of incubation and continuing throughout embryonic development, subviral particles, which resemble viral replication intermediates isolated from infected livers of post-hatch ducklings, appear in the circulation. These particles contain a polymerase activity that utilizes an RNA template to synthesize viral DNA. Our results suggest that certain host functions, which appear during embryonic development, may be required for DHBV replication and assembly. It is possible that in mammals a similar developmental process occurs. The failure to find human hepatitis B virus in the circulation of most babies, born to hepatitis B virus carrier women, in the first few weeks after birth may reflect such a process.     

Nodavirus Colonizes and Replicates in the Testis of Gilthead Seabream and European Sea Bass Modulating Its Immune and Reproductive Functions

Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17β-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases.     

鳜鱼传染性脾肾坏死病毒的胞嘧啶5-甲基转移酶基因结构及其序列分析

对鳜鱼传染性脾肾坏死病毒（infectious spleen and kidney necrosis virus,ISKNV）的胞嘧啶5－甲基转移酶（MTase）基因的结构及序列进行了分析。序列比较分析表明,ISKNV MTase编码区全长684bp,编码长227个氨基酸的蛋白质,推测分子量为25855D。与一些细菌的MTase比较,ISKNV MTase也含有负责转移甲基的4个保守区,但缺乏识别靶序列的保守区。比较ISKNV与其它6种脊椎动物虹彩病毒的MTase序列并建立系统树,ISKNV显著不同于蛙病毒属和淋巴囊肿病毒属。7种脊椎动物虹彩病毒MTase具有高度保守区,可以此设计引物用PCR方法鉴定脊椎动物虹彩病毒。     

Characterization of an iridovirus isolated from the southern mole cricket,Scapteriscus vicinus

A new iridovirus has been detected from diseased southern mole crickets, Scapteriscus acletus, collected in Brazil during the spring of 1986. This icosahedral virus measuring 146 nm (side-side) to 172 nm (apex-apex) has been purified via Ficoll gradient centrifugation and demonstrated to be infectious to 1st instar Scapteriscus vicinus nymphs. The cytopathology of this virus is typical of the pattern documented for other iridovirus isolates. Characterization of the structural polypeptides by SDS-polyacrylamide gel electrophoresis revealed an array of 3 major and 17 minor polypeptides ranging in molecular weight from 15.1 to 152.0 kDa. Electrophoresis in agarose gels of purified DNA revealed a single band of high molecular weight. Analysis of various restriction endonuclease (REN) digests of this DNA demonstrated it to have an approximate molecular weight of 144 kilobase pairs. Based on differences in the polypeptide profile and REN profiles we believe this virus is distinct from previously characterized invertebrate iridovirus isolates.     

鹅圆环病毒感染性克隆的构建及其转染试验

设计带BamHⅠ酶切标记位点的引物,PCR扩增鹅圆环病毒(Goose circovirus,GoCV)全长基因组,将2个基因组顺式连接插入到pGEM-T Easy载体中,获得GoCV全长基因组头尾串联二聚体感染性克隆质粒pGEMT-2GoCV。EcoRⅠ酶切线性化pGEMT-2GoCV,与脂质体混合转染GoCV阴性鹅胚和雏鹅,常规PCR检测发现GoCV在转染鹅体内增殖,鹅胚转染组于孵出第2周和第4周检出血清阳性,且其中一个个体于4周龄扑杀时检出法氏囊阳性,雏鹅转染组于转染后2周检出血清阳性。试验进一步对扩增片段进行了BamHⅠ标记位点的检测,并应用GoCV实时荧光定量PCR方法对转染阳性样品进行了定量,结果显示阳性法氏囊组织中病毒含量为1.57×106拷贝/mg,阳性血清含病毒拷贝数在3.52×104～5.92×105拷贝/μL。综上,本试验构建的GoCV全长基因组头尾串联二聚体感染性克隆DNA可以转染鹅胚和雏鹅并增殖出带标记的GoCV克隆。     

鳜鱼传染性脾肾坏死病毒(ISKNV)PCR检测方法的建立及虹彩病毒新证据

利用真鲷虹彩病毒（ＲＳＩＶ）核苷酸还原酶小亚单位（ＲＮＲＳ）基因保守区设计的一对引物,建立了鳜鱼传染性脾肾坏死病毒（ＩＳＫＮＶ）特异的ＰＣＲ检测体系。运用该体系检测ＩＳＫＮＶ,具有简便、快速、敏感、特异等特点,为诊断与预防ＩＳＫＮＶ提供了一个重要的手段。通过对ＰＣＲ产物的克隆与序列分析,发现ＩＳＫＮＶ ＰＣＲ扩增产物与ＲＳＩＶ ＲＮＲＳ基因相应序列的同源性很高,达到９２．５％,进一步证明ＩＳＫＮＶ     

Unhatched eggs: methods for discriminating between infertility and early embryo mortality

Unhatched eggs are a common phenomenon in birds and are often referred to as being 'infertile', which (confusingly) can mean at least two things: (1) that the ovum has not been fertilized or (2) that the embryo has died during development. These two broad categories of hatching failure can be difficult to distinguish, particularly in the early stages of embryo development. We describe methods to distinguish between infertility (due to insufficient sperm) and early embryo mortality in passerine eggs using the Zebra Finch Taeniopygia guttata as a model. We also describe how we successfully adapted these methods for use on eggs from a wild species, the Tree Sparrow Passer montanus , collected after the incubation period, and show that sperm can be visualized on the perivitelline layer of unhatched eggs even several weeks after laying.     

Phylogenetic analysis of the major capsid protein gene of iridovirus isolates from cultured flounders Paralichthys olivaceus in Korea

In 2003, 13 isolates of iridovirus were obtained from cultured flounders Paralichthys olivaceus during epizootics in Korea. The full open reading frames (ORFs) encoding the major capsid protein (MCP) (1362 bp) from the 13 flounder iridoviruses (FLIVs) were sequenced and the deduced amino acid sequences were phylogenetically analyzed. Phylogenetic analysis of the MCP revealed that all 13 FLIVs were the same species as rock bream iridovirus (RBIV), red sea bream iridovirus (RSIV), and infectious spleen and kidney necrosis virus (ISKNV), and were grouped into an unknown genus which was different from the 2 genera known to infect fish, Ranavirus and Lymphocystivirus. This is the first report on the isolation and phylogenetic analysis of the iridovirus of unknown genus from flounders during epizootics.     

Electroporation Facilitates Introduction of Reporter Transgenes and Virions into Schistosome Eggs

Background

The schistosome egg represents an attractive developmental stage at which to target transgenes because of the high ratio of germ to somatic cells, because the transgene might be propagated and amplified by infecting snails with the miracidia hatched from treated eggs, and because eggs can be readily obtained from experimentally infected rodents.

Methods/Findings

We investigated the utility of square wave electroporation to deliver transgenes and other macromolecules including fluorescent (Cy3) short interference (si) RNA molecules, messenger RNAs, and virions into eggs of Schistosoma mansoni. First, eggs were incubated in Cy3-labeled siRNA with and without square wave electroporation. Cy3-signals were detected by fluorescence microscopy in eggs and miracidia hatched from treated eggs. Second, electroporation was employed to introduce mRNA encoding firefly luciferase into eggs. Luciferase activity was detected three hours later, whereas luciferase was not evident in eggs soaked in the mRNA. Third, schistosome eggs were exposed to Moloney murine leukemia virus virions (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG). Proviral transgenes were detected by PCR in genomic DNA from miracidia hatched from virion-exposed eggs, indicating the presence of transgenes in larval schistosomes that had been either soaked or electroporated. However, quantitative PCR (qPCR) analysis determined that electroporation of virions resulted in 2–3 times as many copies of provirus in these schistosomes compared to soaking alone. In addition, relative qPCR indicated a copy number for the proviral luciferase transgene of ∼20 copies for 100 copies of a representative single copy endogenous gene (encoding cathepsin D).

Conclusions

Square wave electroporation facilitates introduction of transgenes into the schistosome egg. Electroporation was more effective for the transduction of eggs with pseudotyped MLV than simply soaking the eggs in virions. These findings underscore the potential of targeting the schistosome egg for germ line transgenesis.     

Is water uptake by reptilian eggs regulated by physiological processes of embryos or a passive hydraulic response to developmental environments?

Moisture availability is critical for successful embryonic development in many organisms. In most oviparous reptiles, for example, water exchange between eggs and the surrounding environment can have substantial fitness consequences, but regulation of this process is unclear. Here, we evaluate whether water uptake by eggs of the lizard Anolis sagrei is regulated by the presence of a live embryo or is a passive hydraulic response to substrate moisture conditions. Many eggs laid in our captive colony were infertile or contained embryos that died during early stages of development, yet these 'dead' eggs continued to gain mass similar to that of 'live' eggs at least during the first half of incubation. Our results suggest that water uptake by eggs is largely a passive hydraulic process during the first half of incubation, but active regulation by embryos may be necessary during latter stages. Maternal effects (e.g., deposition of salts into yolk) could influence this passive process during early incubation.     

Persistence of betanodavirus in Barramundi brain (BB) cell line involves the induction of Interferon response

The BB cell line derived from the brain tissue of a barramundi (Lates calcarifer) that survived nervous necrosis virus (NNV) infection is persistently infected with NNV. To elucidate whether interferon (IFN) plays a role in the mechanism of NNV-persistent infection in BB cell line, a virus-negative control cell line was obtained by treating BB cells with NNV-specific rabbit antiserum for 5 subcultures. After the treatment, NNV titer or RNA or capsid protein was no longer detected in the cured BB (cBB) cells. Expression of Mx gene, encoding a type I IFN-inducible antiviral protein, was found in BB cells and cBB cells following NNV infection, but not in NNV-free cBB cells. Moreover, expression of Mx gene and antiviral activity against NNV were induced in cBB cells by the treatment with MAb-neutralized BB cell supernatant. Furthermore, NNV persistent infection was induced again in cBB cell culture if multiplicity of infection (MOI) was low (< or = 1). These experimental results indicated that IFN-like cytokines existed in the culture supernatant of BB cells, and IFN-induced response played an important role in protecting the majority of cells from virus lytic infection and regulating NNV persistence in the BB cell line.     

Glucocorticoid metabolism in the in ovo environment modulates exposure to maternal corticosterone in Japanese quail embryos (Coturnix japonica)

Maternal effects have gained attention as a method by which mothers may alter the physiological condition and phenotype of their offspring based upon current environmental conditions. The physiological and phenotypic outcomes of glucocorticoid-mediated maternal effects have been extensively studied in a variety of vertebrates; however, the underlying mechanism is currently unclear. Here, we injected tritiated corticosterone into the yolks of freshly laid Japanese quail eggs (Coturnix japonica) and traced its movement and metabolism through the in ovo development period. We found that corticosterone was extensively conjugated throughout the egg by the end of development, and while minimal corticosterone was detected within the embryo during development, accumulation of a conjugated metabolite in the embryo started to occur on day 6 of development. Because no movement and metabolism of corticosterone occurred in infertile eggs, our findings suggest that embryos are not passive recipients of maternal steroids, but instead appear to possess extensive metabolic capabilities, which may modulate their exposure to maternal steroids.     

青岛文昌鱼LIM类基因Bblim同源框区的cDNA克隆、序列分析及其在胚胎发育中的表达(英文)

不同卵裂球发育命运的特化、亦即胚胎细胞的分化是动物胚胎发育的重要特征。多数胚胎细胞尽管形态特征完全一致,却具有完全不同的发育命运。预示着：在这些细胞中存在有决定发育命运的因素———决定子。本工作克隆了青岛文昌鱼ＬＩＭ类同源框基因的同源框片段。目的在于揭示决定子的分子本质。青岛近海采集性成熟的成年青岛文昌鱼,收集未受精卵、受精卵以及各个不同时期的胚胎,液氮冻存备用。分别制备总ＲＮＡ。根据其它动物ＬＩＭ类同源框基因的序列设计引物（Ｔａｂ．１）,连续进行ＲＴＰＣＲ和ＰＣＲ两次扩增。其中,原肠胚来源的第二次ＰＣＲ产物经电泳鉴定（Ｆｉｇ．１）后,酶切、克隆入质粒、测序、将该片段所在的基因命名为Ｂｂｌｉｍ基因,该片段称为Ｂｂｌｉｍ同源框。根据Ｂｂｌｉｍ基因同源框的核苷酸序列推导出其相应的氨基酸序列（Ｆｉｇ．２）,与其它ＬＩＭ类同源框基因进行比较（Ｆｉｇ．３）后,认为：Ｂｂｌｉｍ基因可归入ｌｉｍ３类基因。比较胚胎发育各个不同时期第二次ＰＣＲ产物的含量———即Ｂｂｌｉｍ基因的转录（Ｆｉｇ．４）,提示：该基因可能在受·精·后·和·原·肠·形·成·期·前·后·两个发育阶段起作用。此外,Ｂｂｌｉｍ基因的同源域与海鞘Ｈｒｌｉｍ的     

Consistency of passerine embryo development and the use of embryonic staging in studies of hatching failure

Hatching failure is widespread in birds, and is usually the result of embryo death rather than infertility. Embryo death can result from both intrinsic and extrinsic factors, some of which may vary across the developmental period. Determining the point at which an embryo died during development may therefore help us to understand the underlying cause of death. Here we describe simple criteria that can be used by field ornithologists to establish the developmental stage of dead embryos found in unhatched passerine eggs, and explain how this can be used to estimate the date of embryo death. We compared the pattern of embryo development over the incubation period for three species, the Zebra Finch Taeniopygia guttata, Blue Tit Cyanistes caeruleus and Great Tit Parus major. We also compared rates of Zebra Finch embryo development under artificial and standard (parental) incubation. Embryo development rates were remarkably similar across the three species and between Zebra Finch embryos under artificial and natural incubation conditions. We therefore suggest that the pattern of embryo development in the Zebra Finch may provide a model for other small passerines with similar incubation periods, but acknowledge that further interspecific comparisons are required before this model is considered more widely applicable. By estimating embryo death dates using our approach, ornithologists will be able to determine temporal patterns of embryo mortality in relation to extrinsic environmental conditions. This approach may shed light on how extrinsic factors such as climate and parental behaviour influence embryo survival in wild birds.     

Assembly of the capsid protein of red-spotted grouper nervous necrosis virus during purification,and role of calcium ions in chromatography

Currently virus-like particles (VLPs) are receiving much attention as platforms for next generation vaccines. However, chromatography-based methods for purifying VLPs remain challenging. Unlike traditional methods using density gradient for purifying VLPs, there have been few advances in explaining how assembled particles can be obtained by chromatography. Nervous necrosis virus (NNV) infects over 30 species of fish and leads to large economic losses in the farmed fish industry. Previously we developed a heparin chromatography-based method for purifying red-spotted grouper NNV (RGNNV) VLPs. However it is unclear how the assembled RGNNV VLPs are obtained by this method. It is known that assembly of NNV capsid proteins depends on calcium ions. In the present study, we found that the yield of purified RGNNV capsid protein in heparin chromatography was enhanced when calcium ions were present during binding. Also, it appears that the capsid protein of RGNNV undergoes partial disassembly and reassembly during sample preparation prior to heparin chromatography and the protein finally undergoes assembly during the chromatography. Therefore, our results indicated that heparin-binding affinity of RGNNV capsid protein is linked to its ability for VLP formation. The assembly of RGNNV capsid proteins recombinantly produced is a good model for explaining VLP formation during chromatography-based purification processes.