锦鲤疱疹病毒ORFl基因的克隆、表达及多克隆抗体的制备

为了进行锦鲤疱疹病毒（Koi Herpesvirus Virus,KHV）诊断方法、疫苗研制和蛋白功能研究。通过选择基因保守性极强的ORFl基因作为研究对象,设计1对特异性引物进行PCR克隆。目的基因与表达载体PET构建重组质粒,将重组质粒转化大肠杆菌E．coliBL21（DE3）后诱导表达,再将成功表达的目的蛋白免疫小鼠制备多克隆抗体。PCR产物经电泳显示,所扩增的基因长度774bp,与目的基因长度相符。蛋白表达产物经SDS．PAGE和Western-bolt检测,重组表达质粒K1-PET成功诱导表达,表达蛋白为37．3KD,为包涵体。免疫小鼠获得多克隆抗体,经酶联免疫检测发现其效价达到1：27000。表明表达出的目的蛋白具有免疫原性,实验获得的表达蛋白和多抗血清为KHV的疫苗研制和免疫学检测方法的建立奠定了基础。     

Pharmacokinetics of famciclovir and penciclovir in tears following oral administration of famciclovir to cats: a pilot study

Objective To validate a means of collecting tears from cats, develop an assay for quantifying famciclovir and penciclovir in tears, and to assess famciclovir and penciclovir concentrations and pharmacokinetics in the tears of cats being treated orally with famciclovir for suspected herpetic disease. Animals Seven client‐owned cats. Procedures Cats were treated orally with a median (range) dose of 40 (39–72) mg of famciclovir/kg three times daily for at least 24 h. At various time points following famciclovir administration, tear samples were collected using Schirmer tear test strips. Tear famciclovir and penciclovir concentrations were measured using liquid chromatography‐mass spectrometry, and concentration‐time profiles were analyzed noncompartmentally. The relationship between famciclovir dose and tear penciclovir concentration near its maximum was evaluated using least squares linear regression. Results Maximum tear famciclovir concentration of 0.305 μg/mL occurred at 2.64 h; elimination half‐life was 2.28 h. Maximum tear penciclovir concentration (0.981 μg/mL) occurred 2.25 h following oral administration of famciclovir; elimination half‐life was 2.77 h. A significant positive correlation was noted between famciclovir dose and tear penciclovir concentration at various time points between 0.5 and 3.75 h following drug administration (P = 0.025). Tear penciclovir concentration exceeded the concentration shown to have in vitro efficacy against feline herpesvirus (FHV‐1) (0.304 μg/mL) in about half of samples collected. Conclusions Oral administration of 40 mg of famciclovir/kg to cats resulted in a tear penciclovir concentration‐time profile that approximated the plasma penciclovir concentration‐time profile and frequently achieved a penciclovir concentration at the ocular surface likely to be effective against FHV‐1.     

Bovine herpesvirus type 1 abortions detected by a semi-nested PCR in Brazilian cattle herds

Bovine herpesvirus type 1 (BHV-1) was investigated by a semi-nested polymerase chain reaction (SN-PCR) and by MDBK cell culture virus isolation in organ fragments from 55 aborted fetuses collected from beef and dairy cattle herds with history of reproductive problems in the North of Paraná State, Brazil. A 425 bp amplicon of the BHV-1 glycoprotein D gene was detected in 14 (25.4%) aborted fetuses. BHV-1 was isolated in MDBK cells from the tissue of 5 (9.1%) fetuses. The specificity of positive results was evaluated by Restriction Fragment Length Polymorphism (RFLP) with Bgl I restriction of DNA amplified by SN-PCR, and by virus-neutralization and immunofluorescence with rabbit anti-BHV-1 polyclonal antibodies for virus isolated in cell culture. The results of this work demonstrate the importance of using other diagnostic techniques, like SN-PCR, for BHV-1 detection in organ fragments from aborted fetuses and the high frequency of this virus in reproductive failures in Brazilian cattle herds.     

抗鲤疱疹Ⅱ型病毒卵黄抗体制备及其功能鉴定

为制备抗鲤疱疹Ⅱ型病毒(CyHV-2)的卵黄抗体,探索防治异育银鲫(Carassius auratus gibelio)鳃出血病的新方法和途径,本研究利用原核表达系统产生具免疫原性的重组CyHV-2-ORF72衣壳蛋白,纯化后免疫蛋鸡;二次免疫后采用间接ELISA法抽检免疫蛋的特异性卵黄抗体(IgY)含量,收集抗体效价...     

New detection of Cyprinid herpesvirus 2 associated with mass mortality in colour crucian carp (Carassius auratus), in China

During October 2016, a mass mortality of colour crucian carp (Carassius auratus), which the affected fish were lethargic, inappetence and anoxic, was occurred in a fish farm located in Chengdu, Sichuan province, China. To elucidate the aetiology of this outbreak, histological and electron microscope examination, molecular investigation were conducted. Pathologic examination revealed multi foci necrosis on haematopoietic organs, gills, hearts and pancreas. Transmission electron microscopy observations exhibited sphere herpesvirus‐like particles distributed amongst the tissues of gill, spleen and kidney. Molecular analysis is verified that the causative agent of this outbreak was Cyprinid herpesvirus 2 (CyHV‐2). This report first report CyHV‐2 in colour crucian carp, which increases the concern about damage of CyHV‐2 and its potential role in species.     

Herpesvirus that caused epizootic mortality in 1995 and 1998 in pilchard, Sardinops sagax neopilchardus (Steindachner), in Australia is now endemic

Propagating epizootics due to Pilchard herpesvirus (PHV) occurred in the Australian population of pilchard, Sardinops sagax neopilchardus (Steindachner) (Clupeidae), in 1995 and 1998-99, with up to 60% losses. No mortality events have been evident in the ensuing 7 years, one reason for which could be that PHV is now endemic. During 2004, a survey was conducted to establish if PHV was present in pilchards in Australia. The pilchard is a highly active, pelagic schooling fish which is found in subpopulations, creating difficulties for the conduct of surveys. It occurs in Australian coastal waters and embayments below about 25 degrees S latitude, feeds on plankton and is predated by birds, mammals and larger fish. It reaches sexual maturity at 2 years of age, spawns at sea, enters embayments when about 5 months old and returns to sea when about 1 year old. It may live for 6-9 years, reaching a maximum length of 200 mm. It forms schools and may travel up to 30 km per day. Pilchards aggregate in mobile shoals of fish containing large highly mobile schools, which interact randomly and exchange individuals. Four subpopulations were defined for the purposes of this survey based on differences in biological characteristics: south-eastern Queensland/northern New South Wales (NSW), Victoria/South Australia (SA), south coast Western Australia (SWA) and west coast Western Australia (WWA). Specimens were obtained from the catch of commercial fishermen using random sampling where possible. Polymerase chain reaction (PCR) for the detection of PHV was performed after appraising the suitability of all available tests according to their impact on sample size requirements, total survey costs and logistical constraints. In the analysis, estimates of true prevalence (TP) of infection and 95% confidence limits were adjusted from the apparent prevalence estimates provided by PCR results. Percentage TP of PHV and corresponding 95% confidence intervals for the four subpopulations: NSW, SA, SWA and WWA were thus estimated as 0 (0-1.5), 31 (22-43), 42 (31-55) and 29 (20-41), respectively. PHV is now endemic in Australian populations of pilchard. Implications of the findings for fisheries management are discussed.     

牡蛎疱疹病毒结构蛋白真核表达系统构建及多聚化特性研究

牡蛎疱疹病毒(OsHV-1)在全球范围内导致牡蛎、扇贝与蚶类的大规模死亡,成为双壳贝类养殖产业的重要威胁。为了解OsHV-1的结构与致病机制。本研究利用HEK293t细胞,构建OsHV-1主要核衣壳蛋白（ORF104和ORF33）的真核表达系统,并对ORF104和ORF33潜在相互作用进行检测。实验首先通过特异性PCR扩增技术得到orf 104和orf 33的基因序列,根据其编码蛋白的理化性质、跨膜区与三维结构等生物信息学分析结果,选择pCDNA3.1构建两种基因的重组表达质粒。重组质粒经大肠杆菌扩增、提取后,利用转染试剂Lipo8000?将pCDNA3.1-orf 104与pCDNA3.1-orf 33分别单独或共转染至人胚肾细胞(HEK293t)。然后,将转染后的细胞培养18 h后裂解收集蛋白。最后利用蛋白免疫印迹检测(Western Blot,WB)与负染电镜检测两种目的蛋白的表达情况。结果显示,实验成功构建了OsHV-1衣壳蛋白ORF104和ORF33的重组表达质粒载体,通过真核细胞表达得到大小约为135与35 ku的目的蛋白。研究表明,表达质粒可在真核表达系统中实现蛋白单独转染与共转染,共转染蛋白间可能存在相互作用的趋势并形成多聚体,其中,ORF33自身即可形成分子量不同的多聚体。本研究首次利用真核表达系统开展OsHV-1关键结构蛋白的表达,为进一步开展该病毒结构蛋白功能与互作,以及病毒入侵机制研究奠定基础。     

共表达与牛疱疹病毒1型VP22融合的猪繁殖与呼吸综合征病毒E及M蛋白的重组伪狂犬病毒的构建及其免疫原性观察

为了研制猪繁殖与呼吸综合征病毒（PRRSV）基因工程疫苗，以伪狂犬病毒（PRV）gE基因缺失标志疫苗株TK^-／gE^-／LacZ^＋为病毒载体，通过同源重组，构建了共表达与牛疱疹病毒1型VP22（BHV-1 VP22）融合的PRRSV E及M蛋白的重组伪狂犬病毒（rPRV）TK^-／gE^-／VP22E^＋／VP22M^＋。经PCR、Southern blot、Western blot证实rPRV构建正确，并能表达与BHV-1 VP22融合的PRRSV E及M蛋白。rPRV在IBRS-2、PK-15细胞中的增殖滴度与PRV亲本株相比无显著差异，表明外源基因的插入不影响rPRV增殖。用该rPRV免疫BALB／c小鼠,检测免疫小鼠抗PRRSV中和抗体及脾淋巴细胞增殖反应，并与未融合VP22的单表达PRRSV E蛋白及共表达E及M蛋白的rPRV TK^-／gE^-／E^＋与TK^-／gE^-／E^＋／M^＋进行比较。结果显示TK^-／gE^-／VP22E^＋／VP22M^＋可诱导小鼠产生更好的体液与细胞免疫反应，BHV-1 VP22发挥了佐剂效应。本研究为研制安全、有效的猪繁殖与呼吸综合征-伪狂犬病二价基因工程疫苗奠定了基础。