Immune characterization of long pentraxin 3 in pigs infected with influenza virus

Long pentraxin 3 (PTX3) is a conserved pattern-recognition secreted protein and a host-defence-related component of the humoral innate immune system. The aim of the present study was to characterize swine PTX3 (SwPTX3) protein expression in influenza virus infected pigs. First, we performed in silico studies to evaluate the cross-reactivity of PTX3 human antibodies against SwPTX3. Secondly, we used in vitro analysis to detect SwPTX3 presence in swine bone marrow dendritic cells (SwBMDC) upon stimulation with different agents by Western blot and immunofluorescence. Finally, the levels of SwPTX3 were assessed in experimental infection of pigs with different strains of influenza virus. This is a novel study where the expression of SwPTX3 was evaluated in the context of a pathogen infection. The initial characterization of SwPTX3 in influenza virus infected pigs contributes to understand the role of PTX proteins in the immune response.     

Clarithromycin suppresses induction of monocyte chemoattractant protein-1 and matrix metalloproteinase-9 and improves pathological changes in the lungs and heart of mice infected with influenza A virus

The influenza A virus (IAV)–cytokine–trypsin/matrix metalloproteinase-9 (MMP-9) cycle is one of the important mechanisms of multiple organ failure in severe influenza. Clarithromycin, a macrolide antibiotic, has immune modulatory and anti-inflammatory effects. We analyzed the effects of clarithromycin on the induction of chemokines, cytokines, MMP-9, trypsin, vascular hyper-permeability and inflammatory aggravation in mice with IAV infection. IAV/Puerto Rico/8/34(H1N1) infection increased the levels of monocyte chemoattractant protein-1 (MCP-1) and cytokines in serum, and MMP-9 and trypsin in serum and/or the lungs and heart. Clarithromycin significantly suppressed the induction of serum MCP-1 and MMP-9 and vascular hyperpermeability in these organs in the early phase of infection, but did not suppress the induction of trypsin, IL-6 or IFN-γ. Histopathological examination showed that clarithromycin tended to reduce inflammatory cell accumulation in the lungs and heart. These results suggest that clarithromycin suppresses infection-related inflammation and reduces vascular hyperpermeability by suppressing the induction of MCP-1 and MMP-9.     

Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus

Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1, H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until this moment there are no reports of IAV infection in wild boars or in captive wild boars populations. Herein the occurrence of IAV in captive wild boars with the presence of lung consolidation lesions during slaughter was investigated. Lung samples were screened by RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR. Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using Illumina's genome analyzer platform (MiSeq). The genomic analysis revealed that the HA and NA genes clustered with IAVs of the human lineage and the six internal genes were derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to monitor IAV evolution in Suidae populations.     

猪流感病毒分子生物学研究

猪流感病毒主要引起一种急性高度接触传染性的群发性呼吸道疾病。本文主要论述了猪流感病毒分子生物学特性、基因组结构及其功能等方面的研究。     

流感病毒的核蛋白是一种多功能RNA结合蛋白

流感病毒核蛋白 (NP)的主要功能是使病毒基因组衣壳化 ,以便 RNA转录、复制和病毒子装配。本综述的目的是说明 NP不仅是一种RNA结合的结构蛋白 ,而且作为病毒与宿主细胞之间的一种关键的配体分子而起作用 ,通过病毒和细胞的大分子之间相互作用来发挥其功能 ,包括 RNA、NP、病毒 RNA依赖的 RNA聚合酶和病毒基质蛋白。 NP也与细胞多肽相互作用 ,细胞多肽包括肌动蛋白、细胞核输入和输出装置所需的成分和细胞核 RNA解旋酶     

宿主蛋白ANP32A对流感病毒功能影响的研究进展

流感病毒是一类危害人和动物健康的RNA病毒,其在宿主细胞内的有效复制离不开宿主蛋白酸性核磷蛋白32家族成员A （ANP32A）和病毒RNA聚合酶的协助和支持。病毒RNA聚合酶由3种蛋白PB1、PB2和PA组成,且ANP32A与病毒RNA聚合酶的最强相互作用需要这3种蛋白的共同参与。ANP32A是酸性富含亮氨酸的核磷蛋白32（ANP32）家族成员,其被确认为支持细胞核中病毒RNA聚合酶活性的关键宿主因子,对流感病毒的复制具有重要的作用。ANP32A的物种特异性差异决定了病毒RNA聚合酶的宿主范围：独特的33个氨基酸序列存在于禽类ANP32A （avANP32A）,而在哺乳动物ANP32A中缺乏此氨基酸序列。avANP32A中特有的33个氨基酸序列能增强ANP32A的功能,从而增加禽源特征流感病毒聚合酶活性。禽流感病毒（Avian influenza virus,AIV）不能有效利用较短的ANP32A （即缺乏独特33个氨基酸序列的ANP32A）,因而哺乳动物ANP32A无法支持禽源特征聚合酶活性,然而在人ANP32A （huANP32A）中插入这33个氨基酸能促进其对AIV聚合酶的支持作用。此外,流感病毒的适应性突变也能增强AIV在哺乳动物中的传播力和致病性。AIV适应哺乳动物时往往会发生E627K突变,以增强其在哺乳动物中的复制能力。作者主要介绍了宿主蛋白ANP32A对流感病毒复制、转录的影响和流感病毒发生适应性突变的作用机制,简要论述了ANP32A与聚合酶的相互作用对流感病毒跨物种感染的分子机制。     

A型流感病毒M2蛋白研究进展

M2蛋白是A型流感病毒所特有的一种结构保守的非糖基化的跨膜蛋白,主要在病毒脱壳时酸化病毒粒子的内部环境,在表面血凝素糖蛋白合成过程中作为质子通道调节高尔基体跨膜转移通道的pH.其核苷酸序列高度保守,几乎不发生变异.其25位～43位氨基酸多肽是金刚烷胺类抗流感病毒药物的作用靶位,氨基酸极小变异都可能导致流感病毒抗药性的产生.M2蛋白胞外区域的氨基酸残基多肽(M2e)的特异性抗体可以在感染流感病毒的动物血清中检测出来,将M2e与相关佐剂联合使用可极太的提高机体对流感病毒的免疫力,在病毒感染中起重要作用,是一种潜在的交叉保护性抗原,常被用来探索具有广谱性和持久性的流感"通用疫苗".     

A型流感病毒基质蛋白研究进展

流感病毒基质蛋白 MA包括 M1和 M2两种 ,在病毒进化过程中其结构是稳定的。 A型流感病毒 M1在病毒复制及感染中起关键作用 ,而 M2是一种跨膜蛋白 ,结构很保守。 M2可调节高尔基体跨膜转移通道的 p H梯度 ,对 HA蛋白起稳定性作用。将 M2与乙肝病毒制成融合蛋白 ( M2 HBc)分别免疫小鼠 ,小鼠获得了 90 %～10 0 %的免疫保护力 ,且具有交叉性和持久性。用研制的含 M2基因的 DNA疫苗 ,免疫小鼠产生了 M蛋白特异性的抗体和 CTL反应 ,并对流感病毒攻击具有保护力     

肠道微生物对流感病毒感染小鼠肺损伤调节作用的研究

流感病毒作为一种常见的呼吸道病毒,是人类健康和世界经济的巨大威胁。目前流感治疗遇到病毒高度突变的问题,不断完善已有的控制流感感染方法的同时,也需要开阔视野从宿主反应的角度探索控制流感的新措施,本研究从肠道微生物的角度探索流感造成肺损伤的机制。提前3周将组合抗生素和益生元添加到小鼠饮水中构建不同的肠道微生物环境,小鼠感染流感病毒后运用16S rDNA技术测定结肠微生物组成,蛋白免疫印迹法测定肺部Th17和Treg细胞转录因子RORγT和Foxp3的表达情况,苏木精-伊红染色、荧光定量PCR测定肺损伤状况。结果表明,提前使用组合抗生素和益生元能够改变肠道菌群组成,通过降低肠道菌群数目和增加肠道拟杆菌相对丰度保护肠道,从而反作用于肺部诱导肺Treg细胞分化,抑制Th17细胞分化,改善流感造成的肺损伤。     

Effect of pooling udder skin wipes on the detection of influenza A virus in preweaning pigs

Influenza A virus (IAV) active surveillance in pigs prior to weaning is commonly conducted by collecting individual samples, mostly nasal swabs. Recently, the use of udder skin wipes collected from lactating sows was identified as an effective sampling method to indicate IAV status of suckling piglets prior to weaning. However, there is limited information on the effect of pooling multiple udder wipes on the ability to detect IAV. We evaluated the effect of pooling 3, 5, or 10 udder wipes on the sensitivity of detecting IAV and compared the results with testing the wipes individually. The likelihood of detecting positive udder wipes decreased with pooling when the initial positive cycle threshold value was ≥31.5; pooling of up to 3 samples could be performed without affecting sensitivity significantly. Our results support pooling of udder skin wipes to conduct surveillance of IAV in pigs prior to weaning.     

甲型H1N1与季节性H1N1流感病毒检测基因芯片的制备与初步应用

建立DNA微列阵技术检测甲型H1N1和季节性H1N1流感病毒的方法,进而探讨该方法用于检测临床标本的可行性。通过生物医学数据库甲型H1N1流感病毒及季节性H1N1亚型流感HA与NA基因进行检索和筛选,应用分子生物学软件,进行序列分析、引物及探针设计,并进行验证。试验所设计的探针可以对甲型H1N1流感与季节性H1N1流感病毒进行区分,用该方法检测了长春市CDC送检的6份疑似甲型H1N1流感病毒临床病例,4份阳性,检测结果同实时荧光定量PCR方法一致。结果表明,利用本研究建立的DNA微列阵技术检测甲型H1N1流感病毒方法,特异性和敏感性强,可作为甲型H1N1流感病毒临床标本检测方法。     

Putative human and avian risk factors for avian influenza virus infections in backyard poultry in Egypt

Highly pathogenic influenza A virus subtype H5N1 causes significant poultry mortality in the six countries where it is endemic and can also infect humans. Egypt has reported the third highest number of poultry outbreaks (n = 1084) globally. The objective of this cross-sectional study was to identify putative risk factors for H5N1 infections in backyard poultry in 16 villages in Damietta, El Gharbia, Fayoum, and Menofia governorates from 2010–2012. Cloacal and tracheal swabs and serum samples from domestic (n = 1242) and wild birds (n = 807) were tested for H5N1 via RT-PCR and hemagglutination inhibition, respectively. We measured poultry rearing practices with questionnaires (n = 306 households) and contact rates among domestic and wild bird species with scan sampling. Domestic birds (chickens, ducks, and geese, n = 51) in three governorates tested positive for H5N1 by PCR or serology. A regression model identified a significant correlation between H5N1 in poultry and the practice of disposing of dead poultry and poultry feces in the garbage (F = 15.7, p < 0.0001). In addition, contact between domestic and wild birds was more frequent in villages where we detected H5N1 in backyard flocks (F = 29.5, p < 0.0001).     

A型流感病毒NS1蛋白的结构与功能

A型流感病毒片段8编码的NS1蛋白是病毒复制和传播的重要调节蛋白。它含有3个功能区,即RNA结合区,eIF4GI结合区和效应区。NS1蛋白在A型流感病毒感染细胞中高水平表达,它的主要功能是抑制NF-κB(nuclear factor-κB)活化和IFN(inter-feron)-α/β介导的抗病毒作用,抑制Jun N末端激酶(jun N-terminal kinase,JNK)和AP-1(activating posttranslation)转录因子活化;下调病毒感染细胞凋亡;促进病毒基因表达,抑制宿主mRNA加工和转运。NS1蛋白的深入研究,为今后预防和治疗A型流感病毒寻找突破口。     

Impact of inland waters on highly pathogenic avian influenza outbreaks in neighboring poultry farms in South Korea

BackgroundSince 2003, the H5 highly pathogenic avian influenza (HPAI) subtype has caused massive economic losses in the poultry industry in South Korea. The role of inland water bodies in avian influenza (AI) outbreaks has not been investigated. Identifying water bodies that facilitate risk pathways leading to the incursion of the HPAI virus (HPAIV) into poultry farms is essential for implementing specific precautionary measures to prevent viral transmission.ObjectivesThis matched case-control study (1:4) examined whether inland waters were associated with a higher risk of AI outbreaks in the neighboring poultry farms.MethodsRivers, irrigation canals, lakes, and ponds were considered inland water bodies. The cases and controls were chosen based on the matching criteria. The nearest possible farms located within a radius of 3 km of the case farms were chosen as the control farms. The poultry farms were selected randomly, and two HPAI epidemics (H5N8 [2014–2016] and H5N6 [2016–2017]) were studied. Conditional logistic regression analysis was applied.ResultsStatistical analysis revealed that inland waters near poultry farms were significant risk factors for AI outbreaks. The study speculated that freely wandering wild waterfowl and small animals contaminate areas surrounding poultry farms.ConclusionsPet birds and animals raised alongside poultry birds on farm premises may wander easily to nearby waters, potentially increasing the risk of AI infection in poultry farms. Mechanical transmission of the AI virus occurs when poultry farm workers or visitors come into contact with infected water bodies or their surroundings. To prevent AI outbreaks in the future, poultry farms should adopt strict precautions to avoid contact with nearby water bodies and their surroundings.     

Effect of vitamin E supplementation on lipid peroxidation in blood and lung of influenza virus infected mice

The influenza virus infection (A/Aichi/2/68) was associated with development of oxidative stress in lung and blood of mice, accompanied by an increase in levels of lipid peroxidation products (conjugated dienes and total malondialdehyde) and a decrease in endogenous amounts of natural antioxidant vitamin E. These effects were most pronounced on the 5th day after virus inoculation, in comparison with those on the 7th. Supplementation of mice with exogenous vitamin E before virus inoculation lead to lung and blood protection against lipid peroxidation. A marked decrease in lipid peroxidation products and an increase in vitamin E content was established in blood and lung on the 5th and 7th day after virus inoculation. The stabilizing effect of vitamin E is dose-dependent in blood and dose-independent in lung, and was most pronounced on the 5th day after virus inoculation in comparison with the 7th day.     

Exposure of domestic swine to influenza A viruses in Ghana suggests unidirectional,reverse zoonotic transmission at the human–animal interface

Influenza A viruses (IAVs) have both zoonotic and anthroponotic potential and are of public and veterinary importance. Swine are intermediate hosts and ‘mixing vessels’ for generating reassortants, progenies of which may harbour pandemic propensity. Swine handlers are at the highest risk of becoming infected with IAVs from swine but there is little information on the ecology of IAVs at the human–animal interface in Africa. We analysed and characterized nasal and throat swabs from swine and farmers respectively, for IAVs using RT‐qPCR, from swine farms in the Ashanti region, Ghana. Sera were also analysed for IAVs antibodies and serotyped using ELISA and HI assays. IAV was detected in 1.4% (n = 17/1,200) and 2.0% (n = 2/99) of swine and farmers samples, respectively. Viral subtypes H3N2 and H1N1pdm09 were found in human samples. All virus‐positive swine samples were subtyped as H1N1pdm09 phylogenetically clustering closely with H1N1pdm09 that circulated among humans during the study period. Phenotypic markers that confer sensitivity to Oseltamivir were found. Serological prevalence of IAVs in swine and farmers by ELISA was 3.2% (n = 38/1,200) and 18.2% (n = 18/99), respectively. Human H1N1pdm09 and H3N2 antibodies were found in both swine and farmers sera. Indigenous swine influenza A viruses and/or antibodies were not detected in swine or farmers samples. Majority (98%, n = 147/150) of farmers reported of not wearing surgical mask and few (4%, n = 6) reported to wear gloves when working. Most (n = 74, 87.7%) farmers reported of working on the farm when experiencing influenza‐like illness. Poor husbandry and biosafety practices of farmers could facilitate virus transmission across the human–swine interface. Farmers should be educated on the importance of good farm practices to mitigate influenza transmission at the human–animal interface.     

A型流感病毒PB1-F2蛋白的结构与功能研究进展

PB1-F2蛋白是一种流感病毒蛋白,蛋白通过诱导巨噬细胞凋亡降低宿主的免疫反应.PB1-F2蛋白介导的细胞杀伤作用通过阻碍专职抗原递呈细胞对后天免疫的调节而增加继发细菌感染的可能性,从而使病毒的致病性增加.现就PB1-F2蛋白结构与功能方面的研究进展做一综述.     

动物A型流感病毒RPA-LFD可视化检测方法的建立

为建立动物A型流感病毒(IV)的重组酶聚合酶扩增-侧流层析试纸条(RPA-LFD)检测方法,本研究针对A型IV基质蛋白(M)基因保守序列设计了引物和探针,通过反应条件优化,建立了RPA-LFD检测方法。特异性试验结果显示,该方法检测动物A型IV呈阳性,与新城疫病毒、鸭坦布苏病毒、鸭肝炎病毒、小鹅瘟病毒等无交叉反应,特异性强;反应时间短、敏感性高,可以在35℃,20 min内检测到最低浓度为10拷贝/μL的重组质粒标准品。利用该方法对从化某活禽市场分离的鸭咽拭子样品共50份进行检测,结果3份为阳性,其余均为阴性,与PCR及病毒分离结果一致,准确性高。本研究首次建立了检测动物A型IV的可视化RPA-LFD检测方法,该方法不依赖任何仪器设备,操作简单、快速,可为基层实验室临床快速检测和流行病学研究提供帮助。     

Two different genotypes of H1N2 swine influenza virus isolated in northern China and their pathogenicity in animals

During 2006 and 2007, two swine-origin triple-reassortant influenza A (H1N2) viruses were isolated from pigs in northern China, and the antigenic characteristics of the hemagglutinin protein of the viruses were examined. Genotyping and phylogenetic analyses demonstrated different emergence patterns for the two H1N2 viruses, Sw/Hebei/10/06 and Sw/Tianjin/1/07. Sequences for the other genes encoding the internal proteins were compared with the existing data to determine their origins and establish the likely mechanisms of genetic reassortment. Sw/Hebei/10/06 is an Sw/Indiana/9K035/99-like virus, whereas Sw/Tianjin/1/07 represents a new H1N2 genotype with surface genes of classic swine and human origin and internal genes originating from the Eurasian avian-like swine H1N1 virus. Six-week-old female BALB/c mice infected with the Sw/HeB/10/06 and Sw/TJ/1/07 viruses showed an average weight loss of 12.8% and 8.1%, respectively. Healthy six-week-old pigs were inoculated intranasally with either the Sw/HeB/10/06 or Sw/TJ/1/07 virus. No considerable changes in the clinical presentation were observed post-inoculation in any of the virus-inoculated groups, and the viruses effectively replicated in the nasal cavity and lung tissue. Based on the results, it is possible that the new genotype of the swine H1N2 virus that emerged in China may become widespread in the swine population and pose a potential threat to public health.     

猪流感病毒多表位抗原的小鼠黏膜免疫试验

为探讨经黏膜途径免疫接种猪流感病毒(SIV)多肽疫苗的可行性,选择STV主要结构蛋白血凝素(HA)的T、B抗原表位,将多个表位片段串联,定向克隆到原核表达载体pET-30a(+)中,对串联表达的重组猪流感病毒蛋白SIV-ep1、SW-ep2、SIV-el2进行了小鼠免疫试验。重组蛋白以包涵体形式表达,经洗涤定量后,采用灌胃途径免疫6周龄KM小鼠,检测免疫小鼠抗体及细胞免疫水平。结果发现三免后各免疫组血清IgG抗体与PBS对照组差异极显著(P<0.01);黏膜分泌型IgA抗体,所有免疫蛋白组与PBS组差异均极显著(P<0.01);细胞免疫检测,所有免疫组小鼠外周血T淋巴细胞增殖明显;猪流感病毒血凝抗体检测,SIV-ep1免疫组、SIV-ep2免疫组、SIV-ep1与LT蛋白免疫组抗SIVH1N1亚型HI效价达1:1024,SIV-el2蛋白免疫组HI效价达1:512。结果表明,猪流感病毒重组多表位抗原灌胃免疫小鼠,能产生理想的黏膜免疫抗体和血清抗体。