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.     

Detection of cyprinid herpesvirus-3 DNA in lake plankton

The disease caused by cyprinid herpesvirus-3 (CyHV-3) severely impacts the natural freshwater ecosystem and damages carp and koi farming, however, the pathway of CyHV-3 transmission remains unclear. It is possible that the virus adheres to plankton, which then facilitate viral movement and transmission, and therefore, it is hypothesised that plankton are involved in the disease dynamics. In this study, plankton were collected at eight sites in the Iba-naiko lagoon; we detected and quantified CyHV-3 DNA from plankton samples. The results of the correlation analysis showed a significant positive correlation between CyHV-3 copies and the number of Rotifera, suggesting that CyHV-3 binds to and/or is concentrated by Rotifera. Our results suggest that plankton affect viral ecology in the natural environment.     

Prevalence and characteristics of Cyprinid herpesvirus 3 (CyHV-3) infection in common carp (Cyprinus carpio L.) inhabiting three rivers in Kochi Prefecture,Japan

Cyprinid herpesvirus 3 (CyHV-3) causes lethal disease in common and koi carp. Mortality by CyHV-3 disease has not been reported since 2011 in Kochi Prefecture, Japan. Here, we detected and quantified CyHV-3 in common carp inhabiting three rivers in the prefecture to examine if the carp are carriers of CyHV-3 as a source of infection. CyHV-3 DNA was detected in 16.7% (12/72) of brain samples in Kagami River, 3.9% (3/76) of brain and 3.9% (3/76) of gill samples in Monobe River, and 5.1% (4/79) of brain and 1.3% (1/79) of gill samples in Wajiki River. CyHV-3 genotypes identified in the 23 samples were classified as the J genotype A1 that has been found in Japan. The CyHV-3 DNA load did not differ statistically between sampling months, indicating that CyHV-3 has been silent in common carp, unlike Lake Biwa where the annual reactivation occurs in spring. Taken together, our results represented definitive evidence that seasonal changes in water temperature do not affect CyHV-3 activity in carp. Considering that infectious virus was not isolated from CyHV-3 DNA-positive samples, it was suggested that CyHV-3 establishes a latent infection in carp populations inhabiting Kagami River, Monobe River and Wajiki River. Further, the presence of circular or concatameric CyHV-3 DNA was detected in five of 23 CyHV-3 DNA-positive samples. Common carp inhabiting Lake Biwa were reported previously to harbor linear but not circular CyHV-3 DNA. This difference suggested that the CyHV-3 genome may be circularized for long-term maintenance without active viral replication.     

Transmission of Cyprinid herpesvirus-3 (CyHV-3) from goldfish to naïve common carp by cohabitation

Cyprinid herpesvirus-3 (CyHV-3) has spread worldwide and has had a major impact on koi and common carp production. Previous studies on the host range of the CyHV-3 found that fish species other than koi and common carp are fully resistant to natural virus exposure. Recently, CyHV-3 was detected in goldfish (Carassius auratus auratus) that were in contact with CyHV-3 infected koi. In the present study, a specific RT-PCR product was amplified from the viral thymidine kinase gene in gills, intestine and brain tissues of CyHV-3 infected goldfish. This implied that CyHV-3 replicated in these goldfish. Also, in the presence of a stress factor such as temperature fluctuation, the CyHV-3 infected goldfish transmitted the virus to cohabitated naïve SPF common carp. CyHV-3 DNA was detected in the cohabitated naïve carp tissues by PCR. The results of this study demonstrate that goldfish is a carrier for CyHV-3, permit virus propagation, and disseminate the virus to susceptible carp causing the disease.     

Generation of monoclonal antibodies specific for ORF68 of koi herpesvirus

Outbreaks of koi herpesvirus (KHV) infection in carp are still a serious problem worldwide. KHV is closely related to other two cyprinid herpesviruses, pox herpesvirus (CHV) and haematopoietic necrosis herpesvirus (CyHV-2) in goldfish. In this study, two major KHV antigenic proteins (ORF62 and ORF68) were identified by immunoscreening using a KHV-specific polyclonal antibody, and then monoclonal antibodies were generated for immunodiagnostic studies. After screening hybridoma cells, one mAb against ORF68 (mAb-7C6) was obtained but no mAbs against ORF62. mAb-7C6 specifically reacted with a lysate of KHV-infected koi fin cells (KF-1 cells) but not with lysates of CHV- or CyHV-2-infected KF-1 cells in an immuno-blotting analysis. Similar results were shown in the following tests: (1) a indirect fluorescent antibody test using infected KF-1 cells and (2) an immunohistochemical investigation by fast red stain (infected liver) or FITC detection (infected spleen). These results suggested that mAb-7C6 specifically reacts with KHV ORF68 protein.     

Molecular genetic analysis of Dichelobacter nodosus proteases AprV2/B2, AprV5/B5 and BprV/B in clinical material from European sheep flocks

Dichelobacter nodosus, the etiological agent of ovine footrot, exists both as virulent and as benign strains, which differ in virulence mainly due to subtle differences in the three subtilisin-like proteases AprV2, AprV5 and BprV found in virulent, and AprB2, AprB5 and BprB in benign strains of D. nodosus. Our objective was a molecular genetic epidemiological analysis of the genes of these proteases by direct sequence analysis from clinical material of sheep from herds with and without history of footrot from 4 different European countries. The data reveal the two proteases known as virulent AprV2 and benign AprB2 to correlate fully to the clinical status of the individuals or the footrot history of the herd. In samples taken from affected herds, the aprV2 gene was found as a single allele whereas in samples from unaffected herds several alleles with minor modifications of the aprB2 gene were detected. The different alleles of aprB2 were related to the herds. The aprV5 and aprB5 genes were found in the form of several alleles scattered without distinction between affected and non-affected herds. However, all different alleles of aprV5 and aprB5 encode the same amino acid sequences, indicating the existence of a single protease isoenzyme 5 in both benign and virulent strains. The genes of the basic proteases BprV and BprB also exist as various alleles. However, differences found in samples from affected versus non-affected herds do not reflect the currently known epitopes that are attributed to differences in biochemical activity. The data of the study confirm the prominent role of AprV2 in the virulence of D. nodosus and shed a new light on the presence of the other protease genes and their allelic variants in clinical samples.     

Ingestion of energy, protein and amino acids from pasture by grazing European wild boar (Sus scrofa L.) in a semi-extensive production system

The aim of the study was to determine the apparent consumption of dry matter (DM), gross energy (GE), crude protein (CP) and amino acids (AA) from pasture by European wild boar in a pastoral system. Two pasture-types were used, one consisting predominantly of Lolium perenne L. and the other predominantly of Plantago lanceolata L. The study was conducted in Spring and repeated in Summer. Twelve purebred European Wild Boar of 18.8 ± 0.8 kg (mean ± sem) with nose rings were randomly grouped into six pairs. Each day of the 19-day study, a pair of animals was placed into each of three areas of a pasture strip (1.4 × 6.3 m per area) from each pasture type from 8:30 h until 16:30 h, after which the animals entered a barn and had free access to a commercial diet for 45 min, with each pasture strip being grazed once. Pasture samples were taken on days 4 to 19 from each grazed area pre- and post-grazing and the DM content of these samples was used to calculate DM consumption of the animals. Additional pasture samples were collected and analysed for gross energy, crude protein and amino acids. The wild boar consumed (mean + SEM) 418 ± 72.2 and 210 ± 38.3 g of DM per day in the L. perenne paddock during Spring and Summer, respectively, and 550 ± 85.9 and 226 ± 44.8 g DM per day in the P. lanceolata paddock during Spring and Summer, respectively. The amount of DM, energy, crude protein and amino acids that the animals consumed varied markedly between days, but did not significantly differ in amount between the L. perenne and P. laceolata paddocks. However, the consumptions were significantly lower in Summer than in Spring. It is estimated that the wild boar would have satisfied somewhat less than 90 and 45% of their daily maintenance digestible energy requirements through consumption of pasture when grazing the L. perenne paddock in Spring and Summer, respectively.     

MC3R基因对京海黄鸡屠体性状的遗传效应分析

本研究以MC3R基因为鸡屠体性状的候选基因,旨在探讨其对京海黄鸡屠体性状的影响。运用PCR-SSCP方法结合测序技术,检测了MC3R基因在京海黄鸡群体中的多态性,并采用一般线性模型(GLM)分析基因型与屠体性状的遗传效应。结果显示,MC3R基因CDS序列检测区内,引物MR2、MR3和MR8扩增的片段具有多态性,其中引物MR2和MR3扩增片段均存在2个SNP位点,分别形成AA、AB、AC 3种基因型和EE、EF、FF、EH 4种基因型,引物MR8扩增片段只存在1个SNP位点,形成了KK、KL和LL 3种基因型;检测到的5个SNPs位点分别为CDS区C160A、G192T、C273T、G310A、G762A突变。关联分析结果显示,MR2位点对京海黄鸡各屠体性状(除腹脂质量)均有极显著影响(P0.01);MR3位点各基因型(排除个体数少于3的基因型)屠体性状间差异均不显著(P0.05),推断MR3位点对京海黄鸡屠体性状影响不显著(P0.05);MR8位点对京海黄鸡屠体质量、胸肌质量、腿肌质量、全净膛质量、半净膛质量有极显著影响(P0.01),对活体质量和腹脂质量有显著影响(P0.05)。对MR2和MR8 2个位点的效应进行联合分析,结果显示,2个位点组合基因型对所有屠体性状均有显著或极显著影响(P0.05或P0.01),其中AB/KK基因型组合为有利基因型组合,AA/LL基因型组合为不利基因型组合。因此,本研究可为京海黄鸡分子标记辅助育种提供依据,有利于加快对京海黄鸡屠体性状选择的育种进程,同时通过本研究可推测MC3R基因对家禽的屠体性状具有很大的遗传效应,为家禽育种工作提供理论依据。     

2011—2015年环洞庭湖区H3亚型禽流感病毒的分离鉴定与遗传演化

为了解环洞庭湖地区家禽H3亚型禽流感病毒的感染情况和进化规律,笔者对2011—2015年湖南省环洞庭湖地区的多个活禽市场与散养鸭场分离的31株H3亚型禽流感病毒进行基因测序和进化分析。结果表明:所有病毒的HA裂解位点不含连续的碱性氨基酸,为低致病性禽流感病毒的分子特征;各基因片段进化树显示部分病毒的PA与PB2基因与H5在相同分支,部分病毒的NP、PA、PB1与H7处于相同分支,表明H3可能与H5、H7亚型的禽流感病毒发生了复杂的基因交换;散养家禽分离的部分H3亚型禽流感病毒内部基因与越南、韩国等周边国家野鸟源的H3、H6、H7、H11等亚型同源性较高,可能有相同的进化来源。进一步将分离的病毒进行全基因比对,可划分出21种不同的病毒基因型。环洞庭湖地区H3亚型禽流感病毒基因来源复杂,表现出明显的遗传多样性。     

Hepatitis E Virus of Subtype 3a in a Pig Farm,South‐Eastern France

Hepatitis E virus (HEV) has emerged during the past decade as a causative agent of autochthonous hepatitis and is a clinical concern in Western developed countries. It has been increasingly recognized that pigs are a major reservoir of HEV of genotypes 3 and 4 worldwide and pig‐derived food items represent a potential source of infections by these viruses in humans. Hepatitis E virus RNA testing was performed here on faeces from rectal swabs sampled in 2012 from 50 3‐month‐old farm pigs from the same farm located in south‐eastern France than in a previous work conducted in 2007. Pig HEV sequences corresponding to genomic fragments of ORF2 and ORF1 genes were obtained after RT‐PCR amplification with in‐house protocols. Hepatitis E virus genotype was determined by phylogenetic analysis. Prevalence was similar to that determined 5 years earlier (68% versus 62%). Two robust phylogenetic clusters of HEV subtypes 3a and 3f were identified, and these sequences obtained in 2012 largely differ compared with those obtained in 2007. Notably, HEV sequences obtained in 2012 from a majority (62%) of the infected pigs belonged to subtype 3a, which was not previously described in France, including not being found in any of humans, pigs or wild boars. Further studies are needed to assess the circulation of HEV‐3a in pigs and humans in this country. In addition, along with previous findings, this study supports the need for increased information to the public on the risk of HEV infection through contacts with pigs or consumption of pig‐derived products in France.     

The prevalence and distribution of Mycobacterium bovis infection in European badgers (Meles meles) as determined by enhanced post mortem examination and bacteriological culture

The accurate diagnosis of Mycobacterium bovis infection in badgers is key to understanding the epidemiology of tuberculosis in this species and has significant implications for devising strategies to limit spread of the disease. In this study, badgers (n = 215) in the Republic of Ireland were examined at post mortem and tissues were collected from a range of anatomical locations and pooled into groups for bacterial culture of M. bovis. By assessing confirmed gross visible lesions (VL) alone, infection was detected in 12.1% of badgers. However, by including the results of all culture positive pooled samples, the overall infection prevalence increased significantly to 36.3%. Two-thirds (66.7%) of infected animals had no visible lesions (NVL). While the thoracic cavity (lungs and pulmonary lymph nodes) was found to be the most common site of infection, in a proportion of animals infection was absent from the lungs and draining lymph nodes and was confined to the lymph nodes of the carcase or the head. This may indicate an early extrapulmonary dissemination of infection or alternatively, in the case of the head lymph nodes, a secondary pathogenic pathway involving the lymphoid tissues of the upper respiratory tract (URT).     

Prevalence and age-related infection of Cryptosporidium suis, C. muris and Cryptosporidium pig genotype II in pigs on a farm complex in the Czech Republic

A total of 413 pig faecal samples were collected from pre-weaners (119), starters (131), pre-growers (123) and sows (40) from a farm with a closed breeding system segmented into two breeding complexes and a growing complex in the region of Vysočina, Czech Republic and screened for the presence of Cryptosporidium using staining methods and genotyping (SSU rRNA). Cryptosporidium oocysts were detected by microscopy in the faeces of 21.1% of the samples (87/413). Sequence analyses and RFLP identified C. suis in 44, Cryptosporidium pig genotype II in 23 and C. muris in 2 samples. No mixed infections were found.Pigs under 7 weeks of age were infected with C. suis only. Cryptosporidium pig genotype II was found in animals from 7 weeks of age. No relationship was found between diarrhoea and any Cryptosporidium infection in any of the different age groups (P < 0.05). The pre-weaned pigs shed significantly more Cryptosporidium oocysts than older pigs and it was associated with C. suis infection.     

口蹄疫病毒株China/99 P3区一级结构及与参考毒株的比较分析

用RT－PCR法，以口蹄疫病毒China/99感染的牛舌水泡皮为材料，扩增目的cDNA，与pGEM-T Easy载体连接并转化JM109菌株，再经重组质粒电泳、PCR和EcoR1酶切鉴定。序列测定和分析结果表明，猪源毒在3A基因内缺失10个密码子，与牛源毒的核苷酸和氨基酸序列差异较大。A－G和T－C的转换率较高，而且A－G转换导致氨基酸变异的几率大于T－C转换，它们是影响氨基酸稳定的因素之一。China/99P3区编码产物在第8、120、121、127、132、193、493、501和538位具有特征性氨基酸，可能与该毒株的表型如毒力等有关。3A基因突变率较高，3B、3C和3D较低，3D最为保守，这对维持3C和3D蛋白酶和3B的引物功能至关重要。     

北方三省(区)牛副流感病毒3型的血清学调查

为了解我国北方三省(区)牛副流感病毒3型(BPIV3)的感染状况,从内蒙古、吉林、山西3个省采集牛血清样品482份。采用病毒中和试验方法进行BPIV3抗体检测。结果显示,482份血清中BPIV3阳性共439份,阳性率为91.08%。吉林、内蒙、山西3省区中血清BPIV3阳性率分别97.35%、84.31%、95.15%。研究表明,中国北方三省(区)牛群中普遍存在BPIV3的感染。     

Resistance patterns and detection of aac(3)-IV gene in apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China

The aminoglycoside apramycin has been used widely in animal production in China since 1999. This study was aimed to investigate the resistance pattern of apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China during 2004–2007 and to determine whether resistance to apramycin was mediated by plasmid containing the aac(3)-IV gene and the mode for the transfer of genetic information between bacteria of farm animals and farm workers. Thirty six E. coli isolates of swine, chicken, and human origins, chosen randomly from 318 apramycin-resistant E. coli isolates of six farms in northeastern of China during 2004–2007, were multi-resistant and carried the aac(3)-IV gene encoding resistance to apramycin. Conjugation experiments demonstrated that in all 36 cases, the gene encoding resistance to apramycin was borne on a mobilisable plasmid. Homology analysis of the cloned aac(3)-IV gene with the sequence (accession no. X01385) in GenBank showed 99.3% identity at a nucleotide level, but only with a deletion of guanosine in position 813 of the gene in all 36 cases. The results indicted that resistance to apramycin in these isolates was closely related to aac(3)-IV gene. Therefore, the multi-resistance of E. coli could complicate therapeutic practices for enteric infections in both farm animals and human.     

β‐carotene attenuates lipopolysaccharide‐induced inflammation via inhibition of the NF‐κB,JAK2/STAT3 and JNK/p38 MAPK signaling pathways in macrophages

β‐carotene is one of the most abundant carotenoids, has potential anti‐inflammatory effect, it has been reported that β‐carotene could suppress LPS‐induced inflammatory responses by inhibiting nuclear factor kappa B (NF‐κB) translocation, but the more detailed molecular mechanisms underlying the anti‐inflammatory action of β‐carotene remain to be fully understood. In this study, we investigated the influence of β‐carotene on the activation of JAK2/STAT3, MAPK, and NF‐κB signaling pathway induced by LPS in RAW264.7 cells and peritoneal macrophages. Cells were treated with different concentrations of β‐carotene for 3 hr after LPS treatment for 24 hr. The mRNA expression and the release of IL‐1β, IL‐6, and TNF‐α were evaluated by RT‐PCR and ELISA, and the level of signaling proteins of JAK2/STAT3, MAPK, and NF‐κB signaling pathway were detected by Western blot. The results showed that β‐carotene significantly suppressed (p < 0.05) LPS‐induced release of IL‐1β, IL‐6, and TNF‐α and their mRNA expression. LPS‐induced JAK2/STAT3, IκB/NF‐κB p65, JNK/p38 MAPK signal activation were significantly attenuated (p < 0.05) by β‐carotene in a dose‐dependent manner. In conclusion, β‐carotene could attenuate LPS‐induced inflammation via inhibition of the NF‐κB, JAK2/STAT3, and JNK/p38 MAPK signaling pathways in macrophages.     

Balb/c小鼠线粒体转录终止因子3基因克隆及其组织表达分析

旨在获得Balb/c小鼠线粒体转录终止因子3(MTERF3)基因cDNA的序列特征并进行比较分析,揭示该基因的组织特异性表达规律。应用RT-PCR技术克隆Balb/c小鼠MTERF3基因cDNA编码区序列(CDS),采用MEGA6.0软件构建小鼠MTERF3基因系统进化树,并采用Northern blot、RT-qPCR和Western blot技术研究该基因在小鼠大脑、心、脾、肺、肾、肝、骨骼肌和睾丸等8种不同组织中的特异性表达规律。结果显示,Balb/c小鼠MTERF3基因cDNA的CDS序列大小为1239bp,编码412个氨基酸。小鼠MTERF3基因与大鼠MTERF3基因同源性高达91.1%。MTERF3基因mRNA和蛋白质在Balb/c小鼠各组织中均有不同程度的表达。结果表明,成功克隆了Balb/c小鼠MTERF3基因cDNA的完整编码区序列,并揭示了该基因在各组织器官的表达规律,为进一步研究MTERF3基因在实验动物体内的功能奠定了基础。     

2007年华北地区H3N8亚型马流感病毒的分离与鉴定

2007年10月,华北地区某赛马场的马同时发生了以发烧、流水样鼻汁或脓性分泌物、咳嗽等临床症状为主的疾病,疑似马流行性感冒。采集患病赛马的鼻腔分泌物,发病期和发病后14d血清,经鸡胚接种法分离病毒,并用鸡红细胞血凝抑制试验(HI)、神经氨酸酶抑制试验(NI)、病毒回归试验、血清学检测和基因序列分析对分离的病毒进行了系统鉴定。结果表明分离的毒株(A/equine/Huabei/1/2007(H3N8)为马源H3N8亚型马流感病毒,基因型属于美洲分支。我们通过动物回归感染试验建立起分离毒株的实验感染模型。