The continuous emergence of avian infectious bronchitis virus (IBV) variants poses a critical threat to poultry health and productivity in China. In this study, we conducted comprehensive genetic and antigenic analyses of IBV strains isolated in our laboratory between 2020 and 2024. A total of 94 IBV isolates were sequenced for the S1 gene, revealing widespread nucleotide insertions, deletions, and mutations. Phylogenetic analysis indicated that GI-19 was the predominant genotype (70.21%), followed by GI-13 (21.28%). Recombination analysis using RDP 5.42 identified 14 recombinant strains, primarily GI-13/GI-22 (50%), GI-19/GI-7 (28.6%), and GI-19/GI-22 (21.4%), which were further confirmed using Simplot. Glycosylation analysis revealed that all isolates possessed 14 to18 N-glycosylation sites, whereas only the SDJN3/23 strain contained an O-glycosylation site (position 416). Novel cleavage site motifs (HRRKR, HRHRR, RRFRR) were identified in GI-19 strains, diverging from the canonical HRRRR. The evolutionary rate calculated via BEAST software, was 1.98 × 10-4 substitutions/site/year. Serum neutralization assays demonstrated that GI-19 recombinants exhibited partial one-way cross-protection against GI-1, GI-13, and GI-22 genotypes (titer ≥ 1:32), but reciprocal neutralization was limited. Overall, we systematically characterized the genetic diversity and antigenic evolution of the currently circulating IBV strains in China, emphasizing the critical demand for genotype-specific vaccine development and dynamic surveillance systems to counteract viral immune escape.

Duck circovirus (DuCV) infections cause immunosuppression in ducks, potentially leading to significant economic losses for the duck farming industry. This study investigates the prevalence, genetic characteristics, and evolutionary trends of DuCV in Korea between 2013 and 2022. Samples from 184 farms across seven provinces were analyzed using polymerase chain reaction (PCR). The overall DuCV infection rate was 29.4% (54/184), with Jeollanam-do showing the highest prevalence (37.5%, 15/40). Ducks aged 3-6 weeks were most susceptible to infection, while ducklings younger than one week were rarely infected. Whole-genome sequencing was performed on 24 positive samples with phylogenetic analysis revealing that DuCV-1b is the predominant subtype in Korea (23/24 strains). Notably, a Korean DuCV-1a subtype strain was identified for the first time, showing close genetic relatedness to Chinese DuCV 1a strains. Novel subtype-specific amino acid variations in ORF1 and ORF2 were statistically analyzed and classified. Recombination analysis suggested some Korean DuCV-1b strains may have resulted from recombination events involving strains from different countries. This comprehensive study provides crucial insights into the current prevalence, genetic diversity, and evolutionary dynamics of DuCV in Korea, offering valuable data for developing effective control strategies and understanding the global epidemiology of this economically important avian pathogen.

1 鸡病原检测结果统计分析 2023年1～3月共收到鸡疑似病例样品696份.检测结果显示,以免疫抑制性病原、呼吸道疾病病原、肝损伤性病原和细菌病原检出率相对较高.

Avipoxvirus 282E4 strain was extensively applied into recombinant vaccine vector to prevent other infectious diseases. However, little information on the genomic background, functional and genetic evolutionary of the isolate 282E4 strain was clarified. The results showed that the linear genome of avipoxvirus 282E4 was 308,826 bp, containing 313 open reading frames (ORFs) and 12 new predicted ORFs. The 282E4 strain appears to encode two novel thymidine kinase proteins and two TGF-beta-like proteins that may be associated with the suppression of the host's antiviral response. Avipoxvirus 282E4 also encodes 57 ankyrin repeat proteins and 5 variola B22Rlike proteins, which composed 7% of the avipoxvirus 282E4 genome. GO and KEGG analysis further revealed that 12 ORFs participate in viral transcription process, 7 ORFs may function during DNA repair, replication and biological synthesis, and ORF 208 is involved in the process of virus life cycle. Interestingly, phylogenetic analysis based on concatenated sequences p4b and DNA polymerase of avipoxviruses gene demonstrates that avipoxvirus 282E4 strain is divergent from known FWPV isolates and is similar to shearwater poxvirus (SWPV-1) that belongs to the CNPV-like virus. Sequencing avipoxvirus 282E4 is a significant step to judge the genetic position of avipoxviruses within the larger Poxviridae phylogenetic tree and provide a new insight into the genetic background of avipoxvirus 282E4 and interspecies transmission of poxviruses, meanwhile, explanation of gene function provides theoretical foundation for vaccine design with 282E4 strain as skeleton.

禽流感病毒(Avian influenza virus,AIV)因其具有变异性强、亚型种类多、感染宿主多样性等特点,对畜牧业发展及公共卫生安全具有巨大的影响.目前,传统灭活疫苗在预防禽流感中虽起着重要作用,但仍存在免疫失败、多次接种及易出现不良反应等弊端,因此,研制新型疫苗来弥补传统疫苗的不足非常有必要.纳米颗粒疫苗具有包裹性好、结构稳定、靶向性高和免疫原性强等优点,可作为新型流感病毒疫苗的候选.笔者首先介绍了禽流感难以防控的原因及纳米疫苗的特点,然后对病毒样颗粒疫苗、自组装蛋白疫苗、聚合物纳米颗粒疫苗、无机纳米颗粒疫苗及纳米颗粒的毒性机制方面进行综述,概述了近年来AIV纳米颗粒疫苗的研究进展,并简述了采用不同抗原、不同纳米材料及不同给药方式对免疫效果的影响,结合目前纳米疫苗的研究,预测了未来纳米颗粒疫苗可作为AIV防控的一种新途径,对禽流感纳米疫苗在兽医临床的应用前景进行了分析和展望.

A rabbit rotavirus Z3171 isolate from diarrheic rabbits was identified and sequenced. The genotype constellation of Z3171 is G3-P[22]-I2-R3-C3-M3-A9-N2-T1-E3-H3, which is different from the constellation observed in previously characterized LRV strains. However, the genome of Z3171 differed substantially from those of the rabbit rotavirus strains N5 and Rab1404 in terms of both gene content and gene sequence. Our study suggests that either a reassortment event occurred between human and rabbit rotavirus strains or there are undetected genotypes circulating in the rabbit population. This is the first report of detection of a G3P[22] RVA strain in rabbits in China.

本研究针对病毒基因组5'端非编码区的保守区域设计引物,建立了用于检测鸡传染性腺胃炎相关的圆圈病毒3型(Gyv3)的SYBR GreenⅠ荧光定量PCR方法.该方法具有较好的特异性,敏感性和重复性,对于鸡Gyv3以外的其他常见鸡病毒性病原的DNA或cDNA均无特异性扩增,灵敏度能达到30.76 copies/μL,组内和组间变异系数均不超过2％.对临床样品的检测结果显示,本研究建立的荧光定量PCR方法对鸡Gyv3检出率为12.28％.

Abstract Children and multiple species of domestic animals are susceptible to infection of group A rotaviruses (RVAs) that causes acute gastroenteritis. We isolated an RVA (Z3171) from diarrheic rabbits and characterized its whole-length genome and pathogenicity in rabbits. The genotype constellation of Z3171 was as follows: G3; P[22]; I2; R3; C3; M3; A9; N2; T1; E3; H3,which is different from the constellation observed in previously characterized LRV strains. The result showed that the NSP3 gene of Z3171 belonged to the T1 genotype and shared 96.2% nucleotide identities with other T1 human strain LL4260 circulating in the regions. However the genome of Z3171 differed substantially from that of the other rabbit rotavirus strains, N5 and Rab1404, in terms of both gene content and gene sequence, particularly the NSP5 gene. Our study shows a reassortment event between human and rabbit rotavirus strains, or there may be undetermined gene clusters circulating in the rabbit population. The isolated strain can cause diarrhea in Belgian rabbits in animal regression test. The infection rate was 100%, and the incidence rate was 80%. Further, we report the detection of G3P[22] RVA strain in rabbits from China for the first time.

鸽子呼吸道感染性疾病,通常是一种多病原微生物诱发的混合感染,是鸽舍中常见的疾病. 1 发病原因 细菌、病毒、支原体、衣原体、寄生虫等多种病原引起,其中以鸽毛滴虫、支原体、衣原体感染最为普遍,平时他们潜伏在健康鸽体内,呈菌群平衡状态,但遇到紧迫、疲劳、气候恶劣、抵抗力减弱、大量微生物入侵时,便会单一或混合发病.

兔轮状病毒(lapine rotavirus,LaRV)和兔肠冠状病毒(rabbit enteric coronavirus,RECV)是引起兔腹泻的重要病原,2种病毒引起的仔兔腹泻症状极为相似.为建立能同时鉴别LaRV、RECV的方法,根据LaRV和RECV基因组的保守区段设计2对特异性引物,预期特异性扩增LaRV和RECV的片段大小分别为291 bp和163 bp,经优化反应条件,成功建立了 LaRV和RECV的双重RT-PCR检测方法.特异性检测结果显示,能特异性的扩增出LaRV和RECV的目的条带,与兔瘟、大肠杆菌、魏氏梭菌、沙门菌、泰泽菌、巴氏杆菌、金黄色葡萄球菌、绿脓杆菌、链球菌、支气管败血波氏杆菌均无交叉反应;LaRV和RECV的最低检出限分别为2.73×105,1.22×104拷贝/μL,在相同条件下重复可获得一致的结果.对采自山东部分地区的107份兔腹泻样品的检测结果显示,LaRV和RECV的阳性率分别为10.28％和7.48％,两者与单一 RT-PCR检测结果相一致.结果表明,所建立的双重RT-PCR方法能够快速、准确的对LaRV和RECV单一或混合感染的样品进行检测,为LaRV和RECV的鉴别诊断和流行病学调查提供新的技术手段.

Outbreaks of duck Tembusu virus (DTMUV) have caused serious economic losses in China since 2010. In this study, an infectious clone of the DTMUV BZ-2010strain, isolated from layer cherry duck in China, was constructed using the bacterium-free infectious subgenomic-amplicons method. The subgenomic-amplicons of the human cytomegalovirus promoter (pCMV) at the 5' terminus of the first DNA fragment, the entire genome of DTMUV, and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40pA) at the 3' terminus of the last DNA fragment were synthesized and amplified by PCR in three DNA fragments. The pCMV and HDR/SV40pA were used to drive the viral RNA transcription and generate a full-length RNA transcript of the virus, and were found to be effective in reassembling DTMUV in duck embryo fibroblast cells. The RNA transcripts from the infection clone were infectious in duck embryo fibroblast cells, generating the reconstituted DTMUV. This study provided a valuable reverse genetic tool for the further study DTMUV pathogenesis.

To study the pathogenicity of new duck reovirus (NDRV) to chickens, eighty 3-day-old SPF chickens were equally divided into two groups. The experimental group was inoculated with a NDRV challenge strain of 100 μL (10 –5.00 ELD 50 /0.1 mL) by the subcutaneous (s.c.) route, and the control group was inoculated with 100 μL of sterile phosphate-buffered saline (PBS) by the same route. In the experimental group, chickens exhibited introflexion of claws, performing of splits, stunting syndrome, weight loss and death. Gross lesions such as enlargement and yellowish-white focal necroses were observed in the liver and spleen. Microscopic changes were typical including varying degrees of hepatocyte steatosis and necrosis, splenic lymphocyte necrosis, interstitial pneumonia. Viral loads were detected in lung, liver, heart, spleen, duodenum, burse and kidney. The liver and spleen viral loads remained a much higher level and maintained for a longer time, suggesting that these tissues might be the target organs. In summary, NDRV can cause systemic infections and death in chickens, which indicated that chickens may be infected by NDRV in poultry production.

本研究从发病鹌鹑体内分离出1株支原体,通过菌落形态、pvpA基因的PCR扩增与测序分析,鉴定为鸡毒支原体.采用6种抗菌药物测其最小抑菌浓度(MIC),发现该分离株对泰妙菌素最敏感,MIC达0.00625μg/mL;其次为酒石酸泰乐菌素、盐酸强力霉素,对大观霉素、庆大霉素、卡那霉素的敏感性较差.

雏鸭沙门氏菌病是由沙门氏菌感染引起的一种急性流行性传染性疾病,具有很高的致死率.某种鸭场临床上出现雏鸭零星死亡,采食量下降,剖检可见浆膜炎、包心包肝、肝脏肿大出血、脾脏出血坏死,送检样品经细菌分离、镜检、PCR检测,鉴定为沙门氏菌、大肠杆菌混合感染,根据药敏试验结果,联合使用头孢曲松钠、氟苯尼考治疗,效果显著.

通过对8日龄SPF鸡人工感染鸡传染性支气管炎病毒(IBV) QX株发病,分别在攻毒后3,7,30 d剖杀并采集气管、肺、肾、脾、胸腺、法氏囊制作病理组织切片.攻毒7d后,采集上述器官相同部位进行免疫组织化学法分析.根据IBV-QX株NP基因的保守序列设计引物,构建质粒标准品,建立了IBV-QX Real-time PCR标准曲线,并跟踪检测试验鸡的口腔、泄殖腔排毒情况.此外,观察并记录了2组鸡的临床症状、体质量差异等信息.结果 显示,发病初期(3 d),气管、肺几乎无明显病理变化,其他器官均出现不同程度的炎性反应;临床症状明显期(7 d),所涉及的器官均有不同程度的病理变化;发病后期(30 d),气管纤毛有所恢复,肺、脾炎性反应有所减轻,但肾脏和法氏囊的病变依然严重.免疫组织化学法检测到上述器官均有可见抗原聚集.30 d时被感染鸡的口腔、泄殖腔仍可检出病毒核酸.试验鸡临床症状明显,体质量差异显著.本试验旨在探究具有组织嗜性的IBV-QX毒株对宿主的免疫器官和该病毒的主要靶器官的病理损伤以及被损伤器官的自我修复情况,抗原分布差异,排毒规律等,为后续研究宿主免疫器官发挥天然免疫的相关机理打下基础,也为研发新的疫苗、相关药物的临床药效评价、制定合理的疫苗接种策略提供参考.

从山东某地疑似患肾型鸡传染性支气管炎(IB)的病死鸡的肝、肺、肾脏中分离到1株病毒,经过SPF鸡胚传代接种、血凝试验、鸡胚矮化试验、RT-PCR鉴定及测序分析、动物回归试验,确定为鸡传染性支气管炎病毒,并命名为SD/04/18株.结果 显示,经3％胰蛋白酶处理后,病毒分离株有血凝性,未经处理病毒分离株不具有血凝性;鸡胚矮化试验发现该毒株明显抑制胚胎发育;进化树比对发现,该毒株与我国主要流行的QX株同源性最高,S1基因序列相似性为96.4％,氨基酸序列相似性为95.3％.分离株用SPF鸡胚连续传3代后接种8日龄SPF雏鸡,复制出了与自然病例相同的临床症状,肺、脾、肾、气管等器官出现典型病理变化.免疫组织化学法检测在气管环纤毛、肾小管上皮细胞间隙等部位可见抗原聚集.对被感染试验鸡的口腔、泄殖腔排毒进行跟踪检测,直到试验结束,被感染鸡仍有很高的病毒检出率.结果 表明,最终确定该毒株为肾型鸡传染性支气管炎病毒.

为建立一种检测兔源肺炎克雷伯氏菌(Klebsiella pneumoniae)重组酶聚合酶扩增方法(recombinase polymerase amplification,RPA),根据肺炎克雷伯氏菌的phoE基因保守序列设计引物,扩增片段大小为277 bp,并对反应条件进一步优化,最终建立了适宜于快速准确检测兔源肺炎克雷伯氏菌的重组酶聚合酶等温扩增方法.该方法可特异性检测出兔源肺炎克雷伯氏菌,最低检出细菌量为8.3×101 CFU/mL,灵敏度比传统PCR高100倍.本研究建立的肺炎克雷伯氏菌RPA检测方法特异性强、操作简单,从而为生产中的兔源肺炎克雷伯氏菌现场快速检测提供了一种新方法.

旨在研究肉鸡呼肠孤病毒分离株致病与基因组变异情况.2017年从山东潍坊地区跗关节肿胀、出血严重商品肉鸡中收集病料,进行病毒分离,通过RT-PCR检测、电镜观察对病毒进行鉴定;将分离到的病毒回归商品肉鸡;设计18对引物对分离株全基因组扩增测序,并进行了遗传进化分析;将分离毒株与经典毒株S1133进行血清交叉中和试验.结果表明分离到一株禽呼肠孤病毒(命名为WF17),回归商品肉鸡能完全复制出临床症状,并能从试验鸡中重新分离到该病毒.该分离株基因组完全符合禽呼肠孤病毒基因组结构特点,主要抗原σC蛋白基因与台湾918株最接近,相似性为92.7％,与S1133株的相似性只有55.9％.WF17株与S1133株的抗原相关指数(R值)只有0.19.目前以S1133株为主的商品化疫苗无法对禽呼肠孤病毒变异株产生有效保护.

Reticuloendotheliosis virus (REV) is an important representative avian retrovirus. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of REV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect changes in protein levels in chicken embryo fibroblast cells (CEFs) that were infected with REV or mock infected. In total, 605 cellular proteins were differentially expressed, among which 196, 345, and 286 were differentially expressed in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and immune system processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding, catalytic activity, and enzyme regulator activity. Pathway analysis showed that a total of 143, 167, and 179 pathways, including protein digestion and absorption, focal adhesion, ECM-receptor interaction, cytokine-cytokine receptor interaction, Toll-like receptors, and JAK-STAT signaling, were enriched in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. In conclusion, this study is the first to analyze the protein profile of REV-infected CEFs using an iTRAQ approach. The results of this study provide valuable information for better understanding the host response to REV infection.

Outbreaks of duck Tembusu virus (DTMUV) have caused substantial economic losses in the major duck-producing regions of China since 2010. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of DTMUV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect the protein changes in duck embryo fibroblast cells (DEFs) infected and mock-infected with DTMUV. In total, 434 cellular proteins were differentially expressed, among which 116, 76, and 339 proteins were differentially expressed in the DTMUV-infected DEFs at 12, 24, and 42 hours postinfection, respectively. The Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and cellular organismal processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding and catalytic activity. Some selected proteins that were found to be differentially expressed in DTMUV-infected DEFs were further confirmed by real-time PCR. The results of this study provide valuable insight into DTMUV-host interactions. This could lead to a better understanding of DTMUV infection mechanisms.