Dynamics of Protein 27 of Avian Leukosis Virus and Transforming Growth Factor β2 in Lymphoid Leukosis Susceptible and Resistant Broiler Chicken Breeding Stock

The dynamics of the serum concentration of protein 27 (P27) of avian leukosis virus and transforming growth factor ₂ (TGF-₂) were compared during the period between 29 and 59 weeks of age in two flocks of broiler chicken breeding stock undergoing outbreaks of severe lymphoid leukosis (LL) associated with persistent high mortality (susceptible) and in another two flocks of breeding stock with the presence of avian leukosis virus in association with low mortality due to LL (resistant). The average mean concentration of serum P27 in the LL-susceptible flocks was significantly higher (<0.05) than that in the LL-resistant flocks in six out of seven samplings performed at 5-week intervals, between 29 and 59 weeks of age. The peak in the average rise of serum P27 in the LL-resistant flocks (309 pg/ml) was associated with the highest level of TGF-₂ (1282 pg/ml) among all flocks and at all sampling times. The significance of TGF-₂ in inhibition of lymphoid tumour development is discussed.     

Ethyl acetate fraction of flavonoids from Polygonum hydropiper L. modulates pseudorabies virus-induced inflammation in RAW264.7 cells via the nuclear factor-kappa B and mitogen-activated protein kinase pathways

Pseudorabies virus (PRV) infection leads to severe inflammatory responses and tissue damage, and many natural herbs exhibit protective effects against viral infection by modulating the inflammatory response. An ethyl acetate fraction of flavonoids from Polygonum hydropiper L. (FEA) was prepared through ethanol extraction and ethyl acetate fractional extraction. An inflammatory model was established in RAW264.7 cells with PRV infection to evaluate the anti-inflammatory activity of FEA by measuring cell viability, nitric oxide (NO) production, reactive oxygen species (ROS) release, and mRNA expression of inflammatory factors, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Its functional mechanism was investigated by analyzing the phosphorylation and nuclear translocation of key proteins in the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Our findings indicate that PRV induced inflammatory responses in RAW264.7 cells, and the responses were similar to that in lipopolysaccharide (LPS)-stimulated cells. FEA significantly suppressed NO synthesis and down-regulated both expression and secretion of COX-2, iNOS, and inflammatory cytokines (P<0.05 or P<0.01). FEA also reduced NF-κB p65 translocation into the nucleus and decreased MAPK phosphorylation, indicating that the NF-κB/MAPK signaling pathway may be closely related to the inflammatory response during viral infection. The findings suggested the potential pharmaceutical application of FEA as a natural product that can treat viral infections due to its ability to mitigate inflammatory responses.     

Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China

The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP)assay and to research the recent epidemiology of contagious ecthyma in Jilin Province,China, using the assay. A LAMP assay targeting a highly conserved region of the F1L genewas developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five casesfrom 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 weretested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR.No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth diseasevirus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. Theaverage positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged1–6 months. Our results demonstrated that CEV infection was very widespread in the flocksof Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitivedetection of CEV infection.     

番鸭新病——花肝病的研究：Ⅰ．分离毒的致病必及感染途径试验

番鸭花肝的是近年流行的一种新的番鸭疫病，其特征性病变是死亡番鸭的肝、脾、小肠等部位出现灰白色的坏死点，本文自广东省主要疫区分离到几株病毒，这些病毒在番鸭胚上可继代繁殖，其胚液可使番鸭妇病。不同途径感染试验表明该病毒通过肌肉注射、爪垫部注射、口服，同居感染均可使番鸭发病和死亡，并具有典型病变，分离毒不能使半番鸭，本地鸭，鸡发病。     

鸡贫血病毒荷兰弱毒株基因序列比较研究

用ＣＡ５、ＣＡ６和ＣＡ７、ＣＡ８两对引物对鸡贫血病毒荷兰弱毒株进行ＰＣＲ扩增。将扩增产物１．５ｋｂ和０．８ｋｂ的ＤＮＡ片段分别克隆到ＰＵＣ１１９、ｐＢｌｕｅｓｃｒｉｐｔ载体质粒中,并进行核苷酸序列分析。通过与２６Ｐ４毒株进行核苷酸序列比较发现二者有３２个核苷酸的差异。荷兰和２６Ｐ４两毒株间ＶＰＩ蛋白质同源率为９７％,没有发生特异性变化;ＶＰ３蛋白质同源率为９５％,在Ｉ￣３０个氨基酸之间发生了特异性     

桂林老虎猫瘟热病毒的分离鉴定

我们在作猫细小病毒分子流行病学调查过程中,从桂林送棼的考虑粪便中分离出１株虎细小病毒,并对其进行了系统鉴定,经形态学理化学血清学交叉中和试验、动物感染试验与分子生物学,证明为一株猫瘟热病毒（猫泛白细胞减少症病毒）的强毒。     

猪传染性胃肠炎病毒TaqMan荧光定量RT-PCR检测方法的建立

根据猪传染性胃肠炎病毒和猪肌动蛋白的基因序列设计合成了引物和探针,通过对荧光定量RT-PCR反应条件的优化,建立了TaqMan荧光定量RT-PCR检测TGEV的方法。同时对37份现地病料进行检测并与常规RT-PCR方法、TGEV抗原快速检测试剂盒比较。结果,该方法的检测敏感性达到15.3拷贝/μL,且具有很好的特异性和重复性,而常规RT-PCR方法只能检测到1.53×103拷贝/μL。对37份现地病料的检测结果也表明该法(检出17份)比常规RT-PCR方法(检出12份)和TGEV抗原快速检测试剂盒(免疫层析法,检出10份)的敏感性高。     

Ongoing Activity of Toscana Virus Genotype A and West Nile Virus Lineage 1 Strains in Turkey: A Clinical and Field Survey

Toscana virus (TOSV), West Nile virus (WNV) and tickborne encephalitis virus (TBEV) are among major viral pathogens causing febrile disease and meningitis/encephalitis. The impact of these viruses was investigated at a referral centre in Ankara Province, Central Anatolia in 2012, where previous reports suggested virus circulation but with scarce information on clinical cases and vector activity. Serum and/or cerebrospinal fluid samples from 94 individuals were evaluated, in addition to field‐collected arthropod specimens that included 767 sandflies and 239 mosquitoes. Viral nucleic acids in clinical samples and arthropods were sought via specific and generic nested/real‐time PCRs, and antibody responses in clinical samples were investigated via commercial indirect immunofluorescence tests (IIFTs) and virus neutralization. A WNV antigen assay was also employed for mosquitoes. WNV neuroinvasive disease has been identified in a 63‐year‐old male via RNA detection, and the WNV strain was characterized as lineage 1. TOSV infections were diagnosed in six individuals (6.3%) via RNA or IgM detection. Partial sequences in a 23‐year‐old female, presented with fever and transient pancytopenia, were characterized as TOSV genotype A. Febrile disease with arthralgia and/or peripheral cranial nerve involvement was noted in cases with TOSV infections. Previous WNV and TOSV exposures have been observed in 5.3% and 2.1% of the subjects, respectively. No confirmed TBEV exposure could be identified. Morphological identification of the field‐collected mosquitoes revealed Culex pipiens sensu lato (74.4%), Anopheles maculipennis (20.9%), An. claviger (2.1%) and others. Sandfly species were determined as Phlebotomus papatasi (36.2%), P. halepensis (27.3%), P. major s. l. (19.3%), P. sergenti (8.9%), P. perfiliewi (4.4%), P. simici (2.6%) and others. Viral infections in arthropods could not be demonstrated. TOSV genotype A and WNV lineage 1 activity have been demonstrated as well as serologically proven exposure in patients. Presence of sandfly and mosquito species capable of virus transmission has also been revealed.     

猪细小病毒PCR检测与分离研究

根据猪细小病毒(PPV)结构蛋白VP2基因序列,设计合成1对引物,建立了检测PPV的PCR方法。检测14份临床组织,检出阳性11份,阳性检出率为79.5%。阳性样品扩增产物用EcoRⅠ酶切得到了预期的结果,对PCR产物进行测序,测序结果与已发表的PPV基因核苷酸序列比较,同源性达99%~100%,所编码的氨基酸同源性为93%~100%。从PCR检测阳性的一份病料组织中分离出1株PPV。试验证明,建立的PPVPCR检测方法特异性强、敏感性高,适用于临床样品的快速检测。     

H5N1亚型禽流感病毒NS1基因的原核表达及其ELISA检测方法的建立

采用RT-PCR技术扩增了禽流感病毒（AIV）A／Goose／HLJ／p46／2003（H5N1）的NSl基因，将其克隆于融合蛋白表达载体pMAL-c2X上，转化DH5α大肠埃希氏菌感受态细胞，经BamHⅠ和HindⅢ双酶切鉴定及序列分析，表明筛选到了重组质粒pc2X-NS1。SDS-PAGE电泳结果显示，重组质粒转化TB1大肠埃希氏菌后，经0．3mmol／L的IPTG诱导，融合蛋白MBP-NS1得到大量表达，融合蛋白以可溶形式存在，分子质量约为67ku。Western-blotting检测结果表明，融合蛋白MBP-NS1能够与H5N1亚型AIV活病毒感染康复鸭血清发生特异性反应，而不能够与H5N1亚型AIV灭活疫苗免疫鸭血清发生反应。试验初步建立了以纯化的融合蛋白MBP-NS1为包被抗原的间接ELISA检测方法，为AIV灭活疫苗免疫家禽与AIV感染家禽的鉴别诊断奠定了基础。