IntroductionThe H9N2 subtype is a predominant avian influenza virus (AIV) circulating in Chinese poultry, forming various genotypes (A-W) based on gene segment origins. This study aims to investigate the genotypic distribution and pathogenic characteristics of H9N2 isolates from wild birds and domestic poultry in Yunnan Province, China.MethodsEleven H9N2 strains were isolated from fecal samples of overwintering wild birds and proximate domestic poultry in Yunnan, including four from common cranes (Grus grus), two from bar-headed geese (Anser indicus), and five from domestic poultry (Gallus gallus). Phylogenetic analysis was conducted to determine the genotypes, and representative strains were inoculated into Yunnan mallard ducks to assess pathogenicity.ResultsPhylogenetic analysis revealed that five isolates from domestic birds and one from a bar-headed goose belong to genotype S, while the remaining five isolates from wild birds belong to genotype A. These bird-derived strains possess deletions in the stalk domain of NA protein and the N166D mutation of HA protein, typical of poultry strains. Genotype S H9N2 demonstrated oropharyngeal shedding, while genotype A H9N2 exhibited cloacal shedding and high viral loads in the duodenum. Both strains caused significant pathological injuries, with genotype S inducing more severe damage to the thymus and spleen, while genotype A caused duodenal muscle layer rupture.DiscussionThese findings suggest that at least two genotypes of H9N2 are currently circulating in Yunnan, and Yunnan mallard ducks potentially act as intermediaries in interspecies transmission. These insights highlight the importance of analyzing the current epidemiological transmission characteristics of H9N2 among wild and domestic birds in China.

为了建立禽腺病毒4型(FAdV-4)的环介导等温扩增(LAMP)检测方法,本研究以FAdV-4 hexon基因高度保守的区域设计LAMP引物,并对反应体系和反应条件进行优化,同时评价该方法的特异性、灵敏度及对临床样品的适用性.结果显示,本研究建立的LAMP方法的最佳反应条件为63℃扩增65min;反应体系中内外引物最佳比例为8 ∶1,MgSO4和dNTP浓度分别为60 mmol/L和10mmol/L;与FAdV-10、FAdV-8b、FAdV-11、NDV、IBDV、AIV-H9、IBV、ILTV、ORT、CIAV、ALV等均无交叉反应,具有良好的特异性;本方法可检测的DNA最低浓度为7.5×10-8ng/μL,灵敏度是传统PCR法的100倍.用本研究建立的LAMP法和PCR法对80份临床样品进行检测,结果相符率为90.5％.上述结果表明,本研究建立的FAdV-4 LAMP检测方法具有准确性高、特异性强、灵敏度高和临床应用可行性强的优点,且结果易于判定,便于现场检测,为FAdV-4提供了一种新的现场诊断方法.

The primary flight feathers, secondary flight feathers, tail feathers and down feathers of Grus nigricollis and Grus grus were microscopically imaged, and the morphological data were determined and compared by statistical analysis. The quantitative indexes of morphological structure were statistically analyzed by single factor analysis and sample t test, and the Fisher discriminant function of characteristic indexes was established. The results were showed as following: the length of the barb in the inner web of remiges feathers, the angle between the barb in the inner web of tail feathers and the area of the accessory pinna of a primary flight feather, are remarkably different between G. nigricollis and G. grus in the terms of macrostructure; the number of cilia couples and ventral teeth from distal barbules in secondary flight feathers, the number of ventral teeth in proximal barbules of remiges feathers, the number of hamuli from distal barbules in tail feathers, the distance between nodular and diameters of nodular between the 2 kinds of Grus in the terms of microstructure also showed significantly different;macroscopic and microscopic characteristics of the Grus feathers were quantitatively analyzed, and Fisher discriminant function based on the difference index was constructed. The accuracy rate of using back substitution method to test the discriminant equation is 80.2%. It suggested that Grus of primary and secondary flight feathers and tail feathers of sample classification can be achieved by the number of hamuli, cilia couples, ventral teeth from distal barbules, the number of ventral teeth in proximal barbules, the angle between the barb in the inner web, the length of the barb in the inner web of remiges feathers, the area of the accessory pinna and Length of a feather shaft. It further enriches the identification methods or approaches of rare crane species in China.

The seasonal migration of wild aquatic birds plays a critical role in the maintenance, transmission, and incursion of the avian influenza virus (AIV). AIV surveillance was performed during 2020–2021 in two national nature reserves with abundant wild bird resources in Yunnan, China. Four H5N8 AIVs isolates from the common crane were identified by next-generation sequencing. Phylogenetic analysis demonstrated that all eight gene segments of these H5N8 AIVs belonged to clade 2.3.4.4b high-pathogenic AIV (HPAIV) and shared high nucleotide sequence similarity with the strains isolated in Hubei, China, and Siberia, Russia, in 2020–2021. The H5N8 HPAIVs from common cranes were characterized by both human and avian dual-receptor specificity in the hemagglutinin (HA) protein. Moreover, possessing the substitutions contributes to overcoming transmission barriers of mammalian hosts in polymerase basic 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acid (PA), and exhibiting the long stalk in the neck region of the neuraminidase (NA) protein contributes to adaptation in wild birds. Monitoring AIVs in migratory birds, at stopover sites and in their primary habitats, i.e., breeding or wintering grounds, could provide insight into potential zoonosis caused by AIVs.

Fowl adenoviruses (FAdVs) are distributed worldwide in poultry and incriminated as the etiological agents for several health problems in fowls, and are capable of crossing species barriers between domestic and wild fowls. An FAdV strain was, for the first time, isolated from black-necked crane in this study, and was designated as serotype 4 Fowl aviadenovirus C (abbreviated as BNC2021) according to the phylogenetic analysis of its DNA polymerase and hexon gene. The viral genomic sequence analysis demonstrated that the isolate possessed the ORF deletions that are present in FAdV4 strains circulating in poultry fowls in China and the amino acid mutations associated with viral pathogenicity in the hexon and fiber 2 proteins. A viral challenge experiment with mallard ducks demonstrated systemic viral infection and horizontal transmission. BNC2021 induced the typical clinical signs of hepatitis–hydropericardium syndrome (HHS) with swelling and inflammation in multiple organs and showed significant viral replication in all eight organs tested in the virus-inoculated ducks and their contactees at 6 dpi. The findings highlight the importance of surveillance of FAdVs in wild birds.

为了解禽流感病毒(AIV)在野禽和家禽之间的传播特点,本研究于2020年3月～2021年3月在野生鸟类资源丰富的拉市海国际高原湿地采集到野鸟粪便样品1367份、家禽泄殖腔拭子420份和环境水样20份,共计1807份样品,通过鸡胚接种和RT-PCR分离鉴定出1株斑头雁来源的H9N2 AIV和2株家禽来源的H9N2 AIV.利用Illumina Miseq测序平台获得3株分离病毒的全基因组序列,并构建了其遗传进化树,分析了与流感病毒的传播能力及致病力相关的关键氨基酸位点的特征.遗传进化分析结果显示,野禽源和家禽源AIV具有相同的遗传进化特征:3株H9N2 AIV均属于欧亚谱系h9.4.2.5分支的病毒,其HA、NA基因为Y280-like亚群,PB2、M基因为G1-like亚群,PB1、PA、NP和NS基因为F/98-like亚群,推测3株分离株均为重组病毒,重组模式为中国流行的H9N2 AIV S基因型.氨基酸位点分析结果显示,3株分离病毒HA、NA、PB2、PB1、M1和NS1蛋白的突变位点均与病毒跨种属传播能力和小鼠致病力增强相关;同时野禽源H9N2 AIV同样存在家禽源分离株相似的HA蛋白N166D替换和NA蛋白颈部aa63～aa65位点缺失,但PA蛋白中未发生N383D突变.以上结果表明,本研究中野禽源H9N2 AIV可能源于家禽的传播,病毒在自然宿主野禽中发生了适应性突变.本研究首次对拉市海高原湿地野禽源与家禽源H9N2 AIV的遗传变异特征进行了系统分析,为禽流感的综合防控提供基础数据.

禽腺病毒4型(FAdV-4)是引起心包积液-肝炎综合征(HPS-IBH)的主要病原,主要危害鸡、鸭、鹅等家禽并造成家禽行业严重的经济损失.随着HPS-IBH疫病严重程度的增加,家禽间的传染性也急剧升高,国内外加强对该病的研究及防治.论文阐述了FAdV-4病毒粒子主要结构蛋白六邻体(Hexon)、五邻体(Penton)和纤突(Fiber),以及主要结构蛋白在病毒感染中的不同功能.近年来国内暴发的FAdV-4的基因组序列分析特征表明毒株与印度株同源性极高,并且新毒株存在ORF19缺失.还总结了针对FAdV-4的特异性LAMP、ELISA等病毒检测技术和预防FAdV-4的基因工程疫苗和亚单位疫苗,阐述了FAdV-4的病原学特征,可为综合防控HPS-IBH提供参考.

[目的]分析云南省家禽及野鸟禽腺病毒4型(Fowl adenovirus-4,FAdV-4)流行情况、Hexon基因差异性及致病性,为研究FAdV-4流行及Hexon基因对病毒毒力的影响提供参考.[方法]2020年1月-2021年6月在云南省昆明市、曲靖市、楚雄州、大理州、红河州、玉溪市等家禽养殖密集区采集疑似感染FAdV-4家禽肝脏组织样品158份,候鸟迁徙地采集野鸟新鲜粪便310份,采用PCR技术检测FAdV-4核酸,对获得的代表性阳性样品进行病原分离鉴定,并对分离毒株进行致病性及Hexon基因序列分析.[结果]家禽肝脏组织样品中检出FAdV-4核酸阳性样品19份,阳性率为12.03％(19/158);黑颈鹤粪便样品中检出核酸阳性样品1份,阳性率为0.3％(1/310).从FAdV-4核酸阳性样品中分离到7株FAdV-4,致病性研究结果显示,FAdV-4云南分离株均能致鸡胚发育不良,引起4周龄肉鸡发生心包积液-肝炎综合征,鸡胚半数致死量(ELD50)为10-6.17～10-4.32/mL.7株分离株均聚类于FAdV-C亚群,Hexon蛋白与国内FAdV-4高致病性毒株具有相同的188R、193 R、195 Q特征.分离株感染SPF鸡后,临床症状明显,具有高致病性且高致死率,肝细胞病变明显.[结论]研究初步掌握了云南FAdV-4的流行情况,首次在黑颈鹤粪便中检测到FAdV-4病原核酸,分离获得7株FAdV-4,部分毒株Hexon蛋白存在氨基酸突变.

禽白血病(avian leukosis,AL)是由禽白血病病毒(avian leucosis virus,ALV)引起的一种禽类重要免疫抑制性疾病,主要以垂直方式传播,给我国养禽业造成了严重经济损失.目前控制ALV传播的最有效手段是净化,而检测技术是实现净化的重要技术支撑.本文从病原学、病理学、血清学、分子生物学等方面对ALV检测技术进行综述,概述了不同检测技术的研究进展情况,分析了其优缺点和应用条件.随着新技术的发展和完善,各种ALV检测技术得到了不断优化、改进和发展.本文有助于充分了解现有的ALV检测技术,对日常检测中不同检测技术的综合考量、选择或优化组合具有借鉴意义,为提高ALV检出率,缩短净化时间,提高工作效率提供了技术支撑.

为了探讨瞬时性受体电位通道香草酸受体5、6(TRPV5、TRPV6)在胎盘滋养层(CT)细胞钙转运中的作用.取孕7.5d昆明小鼠胎盘组织分离纯化并鉴定CT细胞,重组慢病毒颗粒TRPV5 siRNA/shRNA/RNAi Lentivirus、TRPV6 siRNA/shRNA/RNAi Lentivirus感染CT细胞,实时荧光定量RT-PCR、Western Blot分别检测转染后细胞内TRPV5/6 mRNA及蛋白表达量,激光共聚焦扫描显微镜经钙离子成像分析细胞内Ca2浓度.结果 显示:与无慢病毒载体感染的空白对照组比较,TRPV5/6 siRNA/shRNA/RNAi Lentivirus感染CT细胞后,TRPV5/6 mRNA及蛋白表达水平均下调(P＜0.05),细胞内钙水平显著性降低(P＜0.05),慢病毒转染的2个组间的细胞钙荧光强度未见显著差异(P＞0.05).结果 表明,TRPV5和TRPV6参与了CT细胞的钙转运,对妊娠期胎盘钙转运具有重要的促进作用.

为了解H5N2亚型禽流感病毒(Avian influenza virus,AIV)的血凝素蛋白(Hemag-glutinin,HA)系统进化特征,借助NCBI influenza virus resource数据库,下载禽源A型流感病毒(Influenza A Virus,IAV)H5N2亚型HA核酸序列,分析毒株的宿主、分离时间等特征,并构建HA蛋白的遗传进化树,分析其系统发生.结果显示:NCBI数据库收录的H5N2 AIV的HA核酸序列1 272条,序列在NCBI发布时间集中于2005-2007年、2011年、2014-2016年;582条序列为野禽来源,宿主主要分布于雁形目、鸡形目和鹆形目的野禽.系统发生分析结果表明:H5N2 AIV的HA蛋白可聚类为亚欧谱系及北美谱系两大分支,前者可分为三个亚系,后者可分为两个亚系.研究结果呈现了H5N2 AIV HA基因在时间及空间上的进化特征,为H5N2 AIV引起的疫病预警和防控提供理论基础.

在哺乳动物妊娠期,母体的钙代谢通过适应性调节满足胎儿对钙的需求,包括肠钙吸收的增强、 肾钙排泄的减少.妊娠钙代谢适应性需要钙离子通路蛋白参与,TRPV5/TRPV6是实现钙代谢调节的重要蛋白.TRPV6高表达于小肠,主要介导肠钙转运,TRPV5与肾脏钙离子重吸收相关,此外TRPV6介导胎盘组织的母体—胎儿钙转运.

在2019年1月 －2019年6月对云南出现呼吸道疫病的57个鸡场进行H9亚型禽流感检测的基础上,选取石林和楚雄2个H9亚型禽流感阳性样品进行病毒分离.从分离的H9N2亚型禽流感病毒感染鸡胚尿囊液中提取总RNA,采用特异性引物经反转录PCR分别扩增 HA和 NA 基因,PCR产物纯化后进行测序.序列比对及系统发育分析结果表明,云南2株 H9N2毒株 H A基因核苷酸序列同源性为94.2％,NA基因核苷酸序列同源性为93.6％,系统进化分析表明云南 H9N2亚型禽流感病毒 H A和 NA 基因均属于欧亚谱系中的类 ADK HKY28097分支(Y280-like),ACKYN12019和ACKYN72019 H A基因之间的同源性为94.3％,与参考毒株ACKJX2448的同源性最高,为95.6％～98.5％,与中国流行的 H9N2代表株和疫苗株同源性较低.HA蛋白333－340位裂解位点为PSRSSR↓GLF,具有低致病性禽流感病毒分子特征,受体结合位点均发生E198T和Q234L的突变,具有人样受体结合特征,在29 、141 、298 、305 、313 、492位氨基酸有6个糖基化位点.ACKYN12019和ACKYN72019 NA基因同源性为93.6％,与Y280-like代表毒株的同源性分别为97.1％～97.5％和93.7％～94.6％,NA蛋白缺失63 、64 、65位氨基酸,在44 、69 、86 、146 、200 、234位氨基酸处存在6个潜在的糖基化位点,NA蛋白红细胞结合(HB)位点分析发现,368－369 、399－403 、432位氨基酸处存在变异.研究结果显示,H9N2亚型禽流感病毒一直处于不断的变异之中,故应加强其监测与防控.