Epidemiological investigation and genome analysis of duck circovirus in Southern China

Duck circovirus (DuCV), a potential immunosuppressive virus, was investigated in Southern China from March 2006 to December 2009 by using a polymerase chain reaction (PCR) based method. In this study, a total of 138 sick or dead duck samples from 18 different farms were examined with an average DuCV infection rate of ∼35%. It was found that ducks between the ages of 40∼60 days were more susceptible to DuCV. There was no evidence showing that the DuCV virus was capable of vertical transmission. Farms with positive PCR results exhibited no regularly apparent clinical abnormalities such as feathering disorders, growth retardation or lower-than-average weight. The complete genomes of 9 strains from Fujian Province and 1 from Zhejiang Province were sequenced and analyzed. The 10 DuCV genomes, compared with others genomes downloaded from GenBank, ranged in size from 1988 to 1996 base pairs, with sequence identities ranging from 83.2% to 99.8%. Phylogenetic analysis based on genome sequences demonstrated that DuCVs can be divided into two distinct genetic genotypes, Group I (the Euro-USA lineage) and Group II (the Taiwan lineage), with approximately 10.0% genetic difference between the two types. Molecular epidemiological data suggest there is no obvious difference among DuCV strains isolated from different geographic locations or different species, including Duck, Muscovy duck, Mule duck, Cheery duck, Mulard duck and Pekin duck.     

PCR detection and sequence analysis of duck circovirus in sick Muscovy ducks

The duck circovirus (DuCV) infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province.Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%～97.5% nucleotide sequence identity to all the four Taiwan isolates (TC1/2002, TC2/2002, TC3/2002, TC4/2002), 82.9% identity to the America (33753-52) isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however,showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.     

Isolation and Genomic Characterization of a Duck-Origin GPV-Related Parvovirus from Cherry Valley Ducklings in China

A newly emerged duck parvovirus, which causes beak atrophy and dwarfism syndrome (BADS) in Cherry Valley ducks, has appeared in Northern China since March 2015. To explore the genetic diversity among waterfowl parvovirus isolates, the complete genome of an identified isolate designated SDLC01 was sequenced and analyzed in the present study. Genomic sequence analysis showed that SDLC01 shared 90.8%–94.6% of nucleotide identity with goose parvovirus (GPV) isolates and 78.6%–81.6% of nucleotide identity with classical Muscovy duck parvovirus (MDPV) isolates. Phylogenetic analysis of 443 nucleotides (nt) of the fragment A showed that SDLC01 was highly similar to a mule duck isolate (strain D146/02) and close to European GPV isolates but separate from Asian GPV isolates. Analysis of the left inverted terminal repeat regions revealed that SDLC01 had two major segments deleted between positions 160–176 and 306–322 nt compared with field GPV and MDPV isolates. Phylogenetic analysis of Rep and VP1 encoded by two major open reading frames of parvoviruses revealed that SDLC01 was distinct from all GPV and MDPV isolates. The viral pathogenicity and genome characterization of SDLC01 suggest that the novel GPV (N-GPV) is the causative agent of BADS and belongs to a distinct GPV-related subgroup. Furthermore, N-GPV sequences were detected in diseased ducks by polymerase chain reaction and viral proliferation was demonstrated in duck embryos and duck embryo fibroblast cells.     

鸭前胰岛素原基因多态性及其与屠体性状的关联分析

以384只北京鸭 (Z2系、Z4系、Z2×Z4杂交系)和樱桃谷鸭为材料, 利用PCR-SSCP结合测序技术, 对前胰岛素原基因外显子2与部分内含子的多态性进行了研究, 并分析对屠体性状的遗传效应。结果发现存在2个单核苷酸突变位点, 即在第179位和第195位分别发生了T→C和C→T的突变。适合性χ2检验结果表明, 北京鸭各品系和樱桃谷鸭均处于Hardy-Weinberg平衡状态(P>0.05)。最小二乘分析SNPs与屠体性状的关系表明, 在北京鸭3个品系中, 基因型 BB 在胴体重、全净膛重和胸肌重上极显著(P<0.01)高于基因型AA和AB, 在腿肌重和皮脂重上极显著(P<0.01)高于基因型AB; 基因型AA在皮脂率和全净膛重上极显著(P<0.01)和显著(P<0.05)高于基因型AB。而对于樱桃谷鸭, 只有AB型在皮脂重和腹脂重上显著(P<0.05)高于基因型AA。研究结果表明, 鸭前胰岛素原基因多态性与鸭的部分屠体性状存在显著相关性, 且B等位基因有利于增加鸭的胴体重和胸肌重。     

Complete genome sequence of a novel flavivirus, duck tembusu virus, isolated from ducks and geese in china

Duck tembusu virus (DTMUV) is an emerging agent that causes a severe disease in ducks. We report herein the first complete genome sequences of duck tembusu virus strains YY5, ZJ-407, and GH-2, isolated from Shaoxing ducks, breeder ducks, and geese, respectively, in China. The genomes of YY5, ZJ-407, and GH-2 are all 10,990 nucleotides (nt) in length and encode a putative polyprotein of 3,426 amino acids. It is flanked by a 5' and a 3' noncoding region (NCR) of 94 and 618 nt, respectively. Knowledge of the whole sequence of DTMUV will be useful for further studies of the mechanisms of virus replication and pathogenesis.     

Identification,genotyping, and molecular evolution analysis of duck circovirus

Duck circovirus (DuCV) is a contagious immunosuppressive virus affecting many duck species, which is responsible for multiple outbreaks in poultry industries worldwide. In this study, the first DuCV isolate GH01 was identified in Sichuan by PCR, which shared a high level of nucleotide identity (81.8–99.4%) with sequences of other DuCV isolates available in GenBank. Comparative phylogenetic and pairwise sequence comparison analyses indicated that DuCV could be divided into two genotypes (DuCV-1 and DuCV-2) and six subtypes (1a, 1b, 1c, 2a, 2b and 2c) based on the complete genome sequence. The results revealed that both DuCV-1 and DuCV-2 had evolved from the same ancestor but undergone divergent evolution. Interestingly, phylogenetic analyses indicated that three isolates were classified into a cluster DuCV-2a using complete DuCV genome sequence and cap gene, except rep gene. Recombination analyses revealed that DuCV-2a arose from recombination between DuCV-1a and DuCV-2b isolates within the rep genes, and the recombination events mainly occur both in non-structural protein coding region and structural protein coding region. In addition, the mechanisms of recombination supporting the genetic variability in DuCV isolates were investigated. Likewise, selective pressure indicated that purifying selection had been a major driving force in maintaining diversity among the DuCV isolates. Because eradicating the virus from commercial ducks is impossible, it is necessary to take effective control measures and implement them throughout the world.     

Comments on duck circovirus (DuCV) genotype definition

A standardised methodology has been used to define genotypes based on pairwise sequence comparisons (PASC). PASC is a widely accepted method in virus taxonomy, which is based on the histogram of pairwised differences among sequences. Recently, Zhang et al. (2013) concluded that the average p-distance of duck circovirus (DuCV) between genotypes 1 and 2 was 0.170, and subtype distance thresholds were 0.032 in DuCV-1 and 0.018 in DuCV-2, respectively. However, there might be some concerns on the methodology application to define the genotype of DuCV. Taking into account the concerns mentioned above, our authors conducted the PASC analyses of 54 capsid gene (ORF2) and genomic sequences including all the sequences from Zhang et al. (2013). Our results confirmed the existence of two DuCV genotypes (1 and 2) and, we suggest that DuCV ORF2 and genomic distance genotype thresholds were 0.061 and 0.038, respectively.     

三株新城疫病毒强毒株的生物学特性及全基因组序列分析

2009～2011年从北方发病鸡群和鸭群中分离出3株新城疫病毒（Newcastle disease virus,NDV）。通过致病性指数测定及交叉血凝抑制试验初步分析了3个毒株的毒力和相互之间的同源性。选取鸡源分离株SDLY01与新城疫疫苗株（LaSota）进行了交叉保护试验,选取鸭源毒株SD03对樱桃谷鸭进行攻毒实验,同时设计引物对3个毒株进行了全基因组测序,并与36株NDV参考株进行了分子进化分析。结果表明3个分离株F蛋白裂解位点的氨基酸序列均为112R-R-Q-K-R-F117符合强毒株的序列特征,并与致病性指数测定结果相符。交叉血凝抑制试验发现3个分离株与疫苗株LaSota 的抗原同源性较低为82.5%～89.4%,两个鸡源分离株间的抗原同源性为90%,而鸭源毒株SD03与鸡源毒株SDSG01同源性为100%。交叉保护试验和攻毒实验结果显示传统的LaSota疫苗能对SDLY01流行株提供100%免疫保护,但第5天仍检测到排毒;鸭源毒株SD03对樱桃谷鸭不致病,但能检出排毒,排毒期最长为5d。全基因组测序与分析表明3个毒株基因组长度均为15192bp,属于基因Ⅶd型毒株,与同期流行的鹅源及鸭源NDV毒株之间全基因组核苷酸序列具有高度的同源性,揭示鸭源、鹅源NDV与鸡源NDV在遗传学和流行病学上密切相关。     

PCK1 expression is correlated with the plasma glucose level in the duck

Phosphoenolpyruvate carboxykinase 1 (soluble) (PCK1) is a key gene in gluconeogenesis and glyceroneogenesis. Although its functions have been extensively studied in mice, bats and humans, little is known in ducks. Here, PCK1 functions were studied using a duck domestication model and a 48‐h fasting experiment. We found PCK1 expression significantly decreased in two breeds of domestic ducks (Jinyun Pockmark ducks and Cherry Valley ducks) as compared with wild ducks (Anas platyrhynchos). Simultaneously, plasma levels of glucose, triglycerides and free fatty acid in domestic ducks were lower than in wild ducks. When compared with fed ducks, the plasma triglyceride level was observed to be significantly decreased, while the glucose and free fatty acid levels remained constant in 48‐h fasting ducks. The expression analysis of gluconeogenic genes revealed that fructose‐1,6‐bisphosphatase genes (FBP1 and FBP2) and the glucose‐6‐phosphatase gene (G6PC2) were not changed, whereas PCK1 was significantly upregulated. In addition, the reported regulators of PCK1, including forkhead box A2 (FOXA2) gene and orphan nuclear receptor NR4A family genes (NR4A1, NR4A2 and NR4A3), exhibited similar expression levels between 48‐h fasting ducks and fed ducks, suggesting that PCK1 is not regulated by these genes in the duck under fasting conditions. In conclusion, PCK1 expression may affect plasma levels of glucose, triglycerides and free fatty acid during the duck domestication process. This work demonstrates for the first time in duck that PCK1 is a key gene in maintaining plasma glucose homeostasis during fasting and that the upregulated expression of PCK1 may be responsible for constant plasma free fatty acid level by the glyceroneogenesis process.     

Complete Genome Sequence Analysis of a Duck Circovirus from Guangxi Pockmark Ducks

We report here the complete genomic sequence of a novel duck circovirus (DuCV) strain, GX1104, isolated from Guangxi pockmark ducks in Guangxi, China. The whole nucleotide sequence had the highest homology (97.2%) with the sequence of strain TC/2002 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY394721.1","term_id":"39546117","term_text":"AY394721.1"}}AY394721.1) and had a low homology (76.8% to 78.6%) with the sequences of other strains isolated from China, Germany, and the United States. This report will help to understand the epidemiology and molecular characteristics of Guangxi pockmark duck circovirus in southern China.     

Discovery of a SNP in exon 7 of the lipoprotein lipase gene and its association with fatness traits in native and Cherry Valley Peking ducks

Lipoprotein lipase (LPL) is a multifunctional protein, playing a major role in the hydrolysis of triglycerides in chylomicrons and very low-density lipoproteins. This study was designed to investigate the effects of variants in exon 7 of the LPL gene on duck growth and fatness traits. Two populations (native and Cherry Valley Peking duck populations) were used in this study. Growth and fatness traits were measured in them. Primers for exon 7 of the LPL gene were designed from chicken genomic and cDNA sequences. Polymorphisms were detected by DNA sequencing, and a PCR-SSCP method was developed to genotype the two populations. The results showed one novel polymorphism: a synonymous alteration in exon 7 of the LPL gene (c.91C>T; EU834120 and EU834121). It was associated with abdominal fat weight and percentage of abdominal fat weight of native Peking duck and with percentage of subcutaneous fat plus skin weight and abdominal fat weight of Cherry Valley Peking duck. The alteration in exon 7 of LPL may be linked with potential major loci or genes affecting some fatness traits.     

鸭脂联素基因单核苷酸多态性检测及群体遗传分析

以昆山麻鸭、樱桃谷鸭、高邮鸭、荆江麻鸭、金定鸭, 山麻鸭、龙白鸭和白羽番鸭8个鸭品种为实验材料, 根据鸭脂联素基因开放阅读框序列设计5对引物, 用PCR-SSCP方法进行单核苷酸多态性分析,并对不同品种群体进行群体遗传学分析。结果发现引物4扩增片段上共存有7个单碱基突变, 第430、457、523处的G-A 、A-G、T-C单碱基突变导致第144、153、175个氨基酸分别由丙氨酸（A）变为苏氨酸（T）、异亮氨酸（I）变为缬氨酸(V)、酪氨酸(Y)变为组氨酸(H); 而C507T, T540C, C576T和C597T 4个单碱基突变为沉默突变。鸭群中表现出AA、AB、AC、BB、BC、CC、DD、DE 8种基因型。8种基因型在8个鸭品种间分布存在极显著的差异(P<0.01)。除金定鸭外, 其他品种均处于Hardy-Weinberg平衡状态。群体遗传分析表明金定鸭的纯合度最高, 高邮鸭最低, 其他各群体的纯合度较相近; 金定鸭为低度多态, 高邮鸭为高度多态, 其他品种为中度多态。表明鸭脂联素基因不同品种中具有丰富的单核苷酸多态性, 可以进一步作为候选基因来分析其与脂肪性状的相关性。     

Complete Genome Sequence of a Duck Hepatitis A Virus Type 3 Identified in Eastern China

We report here the complete genome sequence of a novel duck hepatitis A virus type 3 (DHAV-3) isolated from a dead Cherry Valley duckling in eastern China. The whole genomic nucleotide sequence and polyprotein amino acid sequence of the virus had higher homology with those of Chinese DHAV-3 isolates, medium homology with those of Korean DHAV-3 isolates, and the lowest homology with those of Vietnamese isolate DN2. The result indicated that the genetic evolution of DHAV-3 isolates had obvious geographical features.     

Complete Genome Sequence of Duck Tembusu Virus,Isolated from Muscovy Ducks in Southern China

We report here the complete genomic sequence of the duck Tembusu virus (DTMUV) WJ-1 strain, isolated from Muscovy ducks. This is the first complete genome sequence of DTMUV reported in southern China. Compared with the other strains (TA, GH-2, YY5, and ZJ-407) that were previously found in eastern China, WJ-1 bears a few differences in the nucleotide and amino acid sequences. We found that there are 47 mutations of amino acids encoded by the whole open reading frame (ORF) among these five strains. The whole-genome sequence of DTMUV will help in understanding the epidemiology and molecular characteristics of duck Tembusu virus in southern China.     

Production of chicken progeny (Gallus gallus domesticus) from interspecies germline chimeric duck (Anas domesticus) by primordial germ cell transfer

The present study aimed to investigate the differentiation of chicken (Gallus gallus domesticus) primordial germ cells (PGCs) in duck (Anas domesticus) gonads. Chimeric ducks were produced by transferring chicken PGCs into duck embryos. Transfer of 200 and 400 PGCs resulted in the detection of a total number of 63.0 ± 54.3 and 116.8 ± 47.1 chicken PGCs in the gonads of 7-day-old duck embryos, respectively. The chimeric rate of ducks prior to hatching was 52.9% and 90.9%, respectively. Chicken germ cells were assessed in the gonad of chimeric ducks with chicken-specific DNA probes. Chicken spermatogonia were detected in the seminiferous tubules of duck testis. Chicken oogonia, primitive and primary follicles, and chicken-derived oocytes were also found in the ovaries of chimeric ducks, indicating that chicken PGCs are able to migrate, proliferate, and differentiate in duck ovaries and participate in the progression of duck ovarian folliculogenesis. Chicken DNA was detected using PCR from the semen of chimeric ducks. A total number of 1057 chicken eggs were laid by Barred Rock hens after they were inseminated with chimeric duck semen, of which four chicken offspring hatched and one chicken embryo did not hatch. Female chimeric ducks were inseminated with chicken semen; however, no fertile eggs were obtained. In conclusion, these results demonstrated that chicken PGCs could interact with duck germinal epithelium and complete spermatogenesis and eventually give rise to functional sperm. The PGC-mediated germline chimera technology may provide a novel system for conserving endangered avian species.     

2009年云南省2株肠道病毒71型分离株全基因组序列遗传特性分析

对2009年云南省肠道病毒71型分离株KMM09和KM186-09进行全基因组序列测序,并与我国及其它国家流行的EV71基因型进行比较和进化分析。KMM09和KM186-09基因组长为7 409bp,编码2 193个氨基酸,VP1系统进化分析显示2009年云南分离株属于C4基因型的C4a亚型。在结构区,与其它基因型相比较,C基因型之间的核苷酸和氨基酸的同源性高于其它基因型;而在非结构区,C4与B基因型和CA16原型株G10同源性高于其它C基因亚型。通过RDP3重组软件和blast比对分析,发现EV71C4基因型与B3基因型,与CA16原型株G10的基因组在非结构区存在重组。EV71全基因组序列的比较和分析,对了解引起我国手足口病暴发或流行C4基因亚型EV71毒株的遗传特性具有重要意义。     

Cloned duck hepatitis B virus DNA is infectious in Pekin ducks

Approximately 10% of German-bred Pekin ducks were found to be chronically infected with duck hepatitis B virus (DHBV). The genomes of three German DHBV isolates analyzed were closely related but showed substantial restriction site polymorphism compared with U.S. isolates. We tested the infectivity of three sequence variants of cloned DHBV DNA by injecting them into the liver of virus-free ducklings. Most of these animals injected with double-stranded closed-circular or plasmid-integrated dimer DHBV DNA developed viremia, demonstrating the infectivity of all three cloned DHBV DNA variants. The cloned viruses produced were indistinguishable from those from naturally infected animals, implying that our experimental approach can be used to perform a functional analysis of the DHBV genome.     

Fear and stress reactions in two species of duck and their hybrid

Susceptibility to fear and stress has often been studied in birds using selection experiments. Crosses between different species of the same genus have also been used to study the type of transmission observed for "fixed action patterns." In this experiment we used a cross between two species (male muscovy and female pekin ducks) to study the genetic transmission of various characteristics of fear reactions and adrenal activity. Males of the three genetic types (two parental species and the hybrid) were submitted to tonic immobility and fear of man tests. Blood was collected for corticosterone assay before and after physiological stress or injection of 5 microg/kg of immediate synacthen (ACTH agonist). The muscovy duck showed lower levels of fear reactions than the pekin duck in the majority of behavioral tests (six of eight). Corticosterone levels were also nearly always the lowest in muscovy ducks (five of six). Generally speaking, the muscovy duck appeared to be less fearful and less susceptible to stress than the pekin duck. The mule duck was similar to one parent, midway between the two, or showed heterosis for fear of man at 10 weeks of age (more fearful than the two parents). Corticosterone levels were midway between the two parents in the mule duck with only one exception, i.e., where the pekin duck was dominant. The existence of heterosis for fear is surprising, as this character is classically considered to be submitted to stabilizing selection. The transmission of corticosterone levels and other fear characteristics is, however, compatible with stabilizing selection. The results are discussed in terms of adaptability.     

鸭生长激素基因内含子2、3多态性分析

根据鸭生长激素基因内含子2、3的序列设计5对引物,利用PCR-SSCP方法对北京鸭、西湖野鸭、金定鸭、山麻鸭、荆江鸭、绍兴鸭等6个鸭品种进行了单核苷酸多态性分析, 并检测其多态性。结果共发现8个突变位点, 其中内含子2有7个: 2593处C-T, 2770处G-A, 2813处T-A, 2829处C-A, 2894处C-T, 2896处T-C,3100处C-G; 内含子3有1个: 3270处A-G。统计结果显示, 这8个变异位点的基因型频率分布与品种有关, 在这些基因座的变异水平上, 北京鸭和绍兴鸭表现出了相当的品种保守性, 本研究所检测到的这些基因座可能与鸭的生产性能有关。