马疱疹病毒1型主要毒力基因分析及TK基因缺失载体的构建

为了解新疆马疱疹病毒1型（EHV-1）主要毒力基因遗传进化情况并构建TK基因缺失株，本研究以EHV-1 XJ2015株DNA为模板，对其主要毒力基因TK、gI和gE全长进行克隆、测序及生物信息学分析，并扩增TK基因左右重组臂TKL和TKR，构建质粒pUC-TKLR，将扩增后的增强绿色荧光蛋白（EGFP，含有CMV+polyA）插入pUC-TKLR质粒，构建TK基因缺失打靶质粒。TK、gI和gE基因同源性分析结果显示，XJ2015株与国外EHV-1分离株TK、gI和gE基因同源性均较高，分别为99.8%~100.0%、99.6%~100.0%和99.9%~100.0%；与EHV-3分离株同源性均最低，分别为72.9%、59.4%和62.1%；遗传进化分析显示，3个基因均与国外EHV-1同属于一个遗传进化分支，与EHV-9和EHV-4进化关系较近，但与EHV-3进化关系较远，表明XJ2015毒株与国外EHV-1毒株TK、gI、gE基因核苷酸上差异不明显，没有明显的地域性特征，功能基因保守且进化缓慢，同源基因功能相同或相近；经PCR扩增、酶切、测序及转染鉴定，本试验成功构建了用于TK基因缺失的打靶质粒pUC-TKLR-EGFP。通过对EHV-1主要毒力基因的分析及TK基因缺失打靶载体的构建，为新疆地区马鼻肺炎流行病学调查分析、TK基因缺失株的构建提供理论依据。     

Whole-genome sequence of a novel Chinese cyprinid herpesvirus 3 isolate reveals the existence of a distinct European genotype in East Asia

Cyprinid herpesvirus 3 (CyHV3), also known as koi herpesvirus (KHV), can be subdivided primarily into European and Asian genotypes, which are represented by CyHV3-U or CyHV3-I and CyHV3-J, respectively. In this study, the whole genome sequence of a novel Chinese CyHV3 isolate (GZ11) was determined and annotated. CyHV3-GZ11 genome was found to contain 295,119 nucleotides with 52.9% G/C content, which is highly similar to those of published CyHV3-U, CyHV3-I, and CyHV3-J strains. With reference to CyHV3-U, CyHV3-I, and CyHV3-J, CyHV3-GZ11 was also classified into 164 open reading frames (ORF), which include eight repeated ORFs. On the basis of the 12 alloherpeviruses core genes, results from phylogenetic analysis showed that CyHV3-GZ11 had closer evolutionary relationships with CyHV3-U and CyHV3-I than with CyHV3/KHV-J, which were also supported by genome wide-based single nucleotide substitution analysis and the use of a series of developed molecular markers. This study was the first to reveal the presence of a distinct European CyHV3 genotype in East and Southeast Asia at a whole genome level, which will evoke new insights on exploring the origin, evolution, and epidemiology of the virus.     

Comparison of food selection and growth performance of koi carp, Cyprinus carpio L., and goldfish, Carassius auratus (L.) in mono- and polyculture rearing in tropical ponds

To compare the effect of polyculture against conventional monoculture on ornamental carp production, investigations on food selection and growth performance of koi carp (K), Cyprinus carpio L. and goldfish (G), Carassius auratus (L.) were conducted in a 11‐week rearing experiment in two monoculture (100% K and 100% G) and five polyculture (90% K–10% G, 70% K–30% G, 50% K–50% G, 30% K–70% G and 10% K–90% G) conditions in tropical ponds. There were three replicates for each treatment. Environmental conditions and food availability were similar in all the treatments. Ivlev's electivity index showed that both fish species avoided phytoplankton and preferred cladocerans to other zooplankton groups (copepods and rotifers) in monotypic conditions. However, in the polyculture treatments, the positive electivity of goldfish towards cladocerans reduced significantly (P<0.05), while the percentage of copepods, rotifers and phytoplankton in the gut content increased. No significant differences in weight gain, specific growth rate and deformities were recorded at harvest for koi carp between the different treatments (P>0.05). Even the survival rate of koi carp recorded above 90% in all the treatments. However, the goldfish recorded significantly better weight gain, specific growth rate and survival in monoculture (100% G), compared with the polyculture treatments (P<0.05). Goldfish deformities were lowest (P<0.05) in the monoculture treatment (2.42%). The number of marketable fish above a set size limit of 4 g total weight was significantly higher in the two monoculture treatments, compared with the five polyculture treatments (P<0.05). Keeping in view of the dietary similarities of koi carp and goldfish, and the aggressive nature of koi carp in polyculture, it is suggested to refrain from polyculture of goldfish and koi carp until further documentations relating to optimum stocking density and management of polyculture of ornamental carps are available.     

First molecular detection of canine herpesvirus 1 (CaHV-1) in the Eastern Brazilian Amazon

BackgroundCanine herpesvirus type 1 (CaHV-1) infects dogs and is associated with neonatal deaths and reproductive, ocular, neurological, and respiratory problems. In Brazil, reports of CaHV-1 have been restricted to the southeast and south regions, particularly in municipalities in the state of Rio Grande do Sul.ObjectivesTo assess the presence and variability of CaHV-1 in canine populations in the state of Pará, North Brazil.MethodsBiological samples from 159 dogs from 4 municipalities in the State of Pará were evaluated using polymerase chain reaction and phylogenetic analyses, with the target being the viral enzyme, thymidine kinase.ResultsCaHV-1 was detected in 13 dogs (8.2%), with 2 animals being from the municipality of Santa Bárbara do Pará, 8 from Algodoal Island, 2 from Salinópolis, and one from Capanema. The study sequences revealed 100% identity among themselves and 64% to 100% identity with the other nucleotide sequences from Australia, Brazil, United Kingdom, and United States, including 100% identity with the 2002 isolate from Australia. The 1996 isolate from France was grouped in a branch that was different from the sequence of this study.ConclusionsThis study presents the first molecular detection of CaHV-1 in dogs from the Amazon region in northern Brazil. The nucleotide identity between the strains and cytosine insertion in the sequences isolated in this study suggests at least 2 strains of CaHV-1 circulating in Brazil (Pará and BTU-1).     

牛疱疹病毒Ⅰ型Bartha Nu/67株gD基因的克隆及在大肠杆菌中的表达

用PCR方法从感染牛疱疹病毒Ⅰ型Bartha Nu／67株的MDBK细胞中扩增gD基因，将之克隆到pGEM-T Easy载体。根据对gD基因的结构分析，设计合成3对引物，以获得的克隆质粒为模板，PCR扩增gD胞外域基因，将之分别亚克隆到pGEX-4T-2和pET-32a表达载体中构建成表达质粒pGEX-4T—gDQ、pGEX-4T—gDQB、pGEX-4T—gDHB和pET—gDQ、pET—gDQB、pET—gDHB，利用上述质粒分别转化大肠杆菌BL21和BL21（DE3），IPTG诱导后SDS—PAGE检测，结果pGEX-4T—gDQ、pGEX-4T—gDQB、pGEX-4T—gDHB和pET—gDQ四个表达质粒出现表达且符合预期大小，Western blot表明表达产物均有抗原性。     

患“红肝病”鳗鲡疱疹病毒的分离和鉴定

利用鳗鲡疱疹病毒(HVA)DNA聚合酶基因引物对患病双色鳗鲡和美洲鳗鲡进行PCR检测(PCR检测结果仅有双色鳗鲡),获得约394bp的目的条带,测序表明扩增出的条带为AHV的DNA聚合酶基因片段,与GeneBank公布的HQ992949、GU233800、FJ940765等6个鳗鲡疱疹病毒基因同源性为100%,与GU205167、AF363783鳗鲡疱疹病毒基因同源性为99%。将患病双色鳗鲡、美洲鳗鲡肝脏和肾脏组织的除菌滤液,接种EPC细胞,细胞7d出现CPE,双色鳗鲡株传代至15代,美洲鳗鲡株传代至5代。     

自然感染鲤疱疹病毒Ⅱ的病毒载量研究

通过建立靶向于鲤疱疹病毒Ⅱ(Cyprinid herpesvirus 2,Cy HV-2)胸苷激酶基因(TK基因)的荧光定量PCR法,分别检测了自然感染Cy HV-2的金鱼、鲫样品,并结合人工感染试验,分析了脾和肾组织内Cy HV-2的载量及其引发的组织病理变化。结果表明,自然感染Cy HV-2的金鱼和鲫的病毒拷贝数范围为102~105,人工感染Cy HV-2的异育银鲫在感染后第五天脾和肾组织内病毒的拷贝数高达1010。人工感染条件下鱼体呈现系统性病理损伤,自然感染状态下鱼体脾和肾的病理变化分别表现为细胞嗜性的转变和炎症。     

饲料中不同添加剂组合对锦鲤生长和体色的影响

为研究饲料中不同添加剂组合对锦鲤Cyprinus carpio(初始体质量为195.88 g±0.98 g)生长和体色的影响,并与日本优质锦鲤商品饲料进行增重和增色效果对比,进行了增重和增色两部分试验,其中增重部分以三色锦鲤为研究对象,探讨了试验增重饲料与商品增重饲料对其生长的影响,增色部分以红白锦鲤为研究对象,探讨了试验对比饲料、试验增色饲料和商品增色饲料对其体色的影响,各组均设3个重复,试验周期为60 d,每隔15 d测定锦鲤的生长和体色指标。结果表明:添加L-肉碱、血球蛋白粉、螺旋藻和枯草芽孢杆菌的试验饲料对三色锦鲤的促生长效果与商品增色料无显著性差异(P0.05),但试验饲料具有更低的饲料系数;添加虾青素、螺旋藻和枯草芽孢杆菌的试验饲料对红白锦鲤体色的增艳效果与商品增色料无显著性差异(P0.05);投喂两种试验增色料和商品增色料的锦鲤体表各部位对色素的沉积程度相似。研究表明,采用不同添加剂组合的两种试验饲料与日本优质锦鲤饲料的功效水平相当,能够分别满足对锦鲤快速增重和增色的要求。     

饲料浮萍水平对黄金锦鲤生长性能、消化酶活力及抗氧化能力的影响

为研究浮萍Lemna minor对黄金锦鲤Cyprinus carpio生长性能、抗氧化和消化能力的影响,以初始体质量为(52.99±1.95)g的黄金锦鲤为研究对象,在饲料中分别添加0、3%、6%、9%、13%、14%的浮萍,相应替代同等水平的菜粕,制成6组等氮(粗蛋白质为38.5%~38.6%)、等能(12.2 MJ/kg)的饲料,分别记为D1(对照)、D2、D3、D4、D5、D6饲料组,将540尾健康的黄金锦鲤随机分成6组,每组设3个重复,每个重复放30尾鱼,养殖时间为8周。结果表明:D6饲料组增重率(WGR)、蛋白质效率(PER)、特定生长率(SGR)和肥满度(CF)均显著高于对照组及添加水平为3%的饲料组(P0.05),而其饲料系数(FCR)显著低于上述两组(P0.05);D6饲料组蛋白酶活力与脂肪酶活力显著高于其他饲料组(P0.05),各饲料组间淀粉酶活力则无显著性差异(P0.05);D4与D6饲料组肝胰脏、脾脏、肾脏和血清中的超氧化物歧化酶(SOD)与过氧化氢酶(CAT)活力显著高于其他饲料组(P0.05),且D6饲料组丙二醛(MDA)活力显著最低(P0.05)。研究表明:本试验条件下,饲料中浮萍的添加水平在14%时,有利于黄金锦鲤的生长,并能提高鱼体抗氧化能力与消化酶活力。