ALG5对猪流感病毒复制的影响

在实验室前期利用猪肾细胞（PK-15）CRISPR/Cas9全基因组敲除文库（PK-15-GeCKO）筛选到猪流感病毒（swine influenza virus,SIV）复制相关的宿主因子磷酸十二烷基磷酸酯-葡萄糖基转移酶（ALG5）的前提下,深入研究ALG5与SIV复制的关系。本研究利用CRISPR/Cas9技术构建了包含ALG5基因向导RNA（gRNA）的质粒LentiGuide-puro-ALG5,并通过慢病毒包装感染稳定表达Cas9蛋白的新生猪气管上皮（NPTr）细胞系,采用有限稀释法筛选ALG5基因敲除的单克隆细胞株,通过靶基因组测序及Western blot验证了ALG5基因在NPTr细胞上的敲除水平,通过CCK-8试验验证基因敲除细胞和野生型细胞的细胞活性。结合TCID₅₀和Western blot试验检测了ALG5敲除和过表达对SIV复制的影响。同时,检测了SIV感染NPTr细胞后内源性ALG5蛋白表达量的变化。最后检测了ALG5敲除对猪流感毒株F26复制的影响。结果显示：获得了ALG5敲除的NPTr单克隆细胞系（ΔALG5）,ALG5基因敲除细胞与野生型细胞的细胞活性无显著差异。ALG5基因敲除显著抑制SIV的增殖,过表达促进SIV的增殖。在病毒感染条件下,随着病毒的增殖,ALG5的蛋白表达量上调。ALG5基因敲除后,也可以抑制猪流感毒株F26的复制,无毒株特异性。本研究表明,ALG5是影响猪流感病毒复制过程的一个重要宿主因子,为研究猪流感病毒的复制和致病机制奠定了基础。     

The comparative profile of lymphoid cells and the T and B cell spectratype of germ-free piglets infected with viruses SIV,PRRSV or PCV2

Lymphocyte subsets isolated from germ-free piglets experimentally infected with swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) were studied and the profile of these subsets among these three infections was monitored. Germ-free piglets were used since their response could be directly correlated to the viral infection. Because SIV infections are resolved even by colostrum-deprived neonates whereas PRRSV and PCV2 infections are not, SIV was used as a benchmark for an effectively resolved viral infection. PRRSV caused a large increase in the proportion of lymphocytes at the site of infection and rapid differentiation of B cells leading to a high level of Ig-producing cells but a severe reduction in CD2^—CD21⁺ primed B cells. Unlike SIV and PCV2, PRRSV also caused an increase in terminally differentiated subset of CD2⁺CD8α⁺ γδ cells and polyclonal expansion of major Vβ families suggesting that non-specific helper T cells drive swift B cell activation. Distinct from infections with SIV and PRRSV, PCV2 infection led to the: (a) prevalence of MHC-II⁺ T cytotoxic cells, (b) restriction of the T helper compartment in the respiratory tract, (c) generation of a high proportion of FoxP3⁺ T cells in the blood and (d) selective expansion of IgA and IgE suggesting this virus elicits a mucosal immune response. Our findings suggest that PRRSV and PCV2 may negatively modulate the host immune system by different mechanisms which may explain their persistence.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0091-x) contains supplementary material, which is available to authorized users.     

Infection with Porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs

The early release of cytokines by cells involved in innate immunity is an important host response to intracellular pathogens. Gamma interferon (IFN-gamma) is an important cytokine produced during the early stages of an infection by macrophages, natural killer (NK) cells, and other cell types, and it is also a central cytokine mediator for the induction of cellular or Th1 immunity. To better understand innate and adaptive immune responses after infection with Porcine reproductive and respiratory syndrome virus (PRRSV), we investigated serum IFN-gamma concentrations and the duration of viremia. For 2 strains of atypical PRRSV, IFN-gamma was detectable in swine serum soon after infection and lasted for approximately 3 wk. Serum concentrations of IFN-gamma peaked at about 10 d after inoculation and returned to approximately baseline levels by day 22. However, individual pigs manifested short, sporadic increases in the serum concentration of IFN-gamma from 18 to 50 d after inoculation. Prior vaccination blocked the serum IFN-gamma response associated with homologous virus challenge and altered the kinetics of the response after heterologous challenge. Two other respiratory viruses of pigs, Porcine respiratory coronavirus and Swine influenza virus, do not appear to induce serum IFN-gamma. The early production of IFN-gamma in PRRSV-infected pigs might result from activation of NK cells, a response that is more characteristic of immune pathways stimulated by intracellular bacterial and protozoan infections.     

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.     

猪圆环病毒2型免疫抑制机理研究进展

猪圆环病毒2型(PCV-2)致病的一个主要原因是使感染的个体产生免疫抑制,感染猪体免疫细胞受到不同程度的损伤.PCV-2可以在单核细胞、巨噬细胞等抗原递呈细胞中存在,影响这些细胞对于抗原的递呈.PCV-2也可在T淋巴细胞及B淋巴细胞中增殖,引起机体体液免疫及细胞免疫反应能力的下降.调节免疫的各种细胞因子在感染PCV-2后也有一定程度的改变,PCV-2感染可引起IL-10分泌的增加,PCV-2与猪天然干扰素分泌细胞相互作用,可使这些细胞分泌干扰素的能力下降.PCV-2造成免疫抑制也与病毒ORF3分泌的功能蛋白有一定的关系,ORF3可诱导免疫细胞凋亡,主要是激活caspase-8途径而不是caspase-9途径.     

Expression of myeloperoxidase in swine influenza virus (SIV)-infected neutrophils in lungs from pigs experimentally infected with SIV subtype H1N2

The expression of myeloperoxidase (MPO) was examined in the swine influenza virus (SIV)-infected neutrophils in the lungs of pigs experimentally infected with swine influenza virus (SIV) subtype H1N2 by immunohistochemistry. Five pigs each from the infected and non-infected group were euthanized 1, 3, 5, 7, and 10?days post-inoculation (dpi). Immunohistochemical reactivity was mainly seen in neutrophils. The score for pulmonary histopathological lesions correlated with the score for MPO immunohistochemical reactivity (r ( s )?=?0.962, P?相似文献   

JAK/STAT途径对家蚕杆状病毒感染应答的初探

为了探讨家蚕抗病毒感染的免疫应答途径,通过非转座子介导将家蚕信号转导子及转录激活因子(STAT)基因表达盒(BmSTAT)导入家蚕卵巢培养细胞(BmN),经吉欧霉素(zeocin)筛选获得过表达BmSTAT的稳定转化BmN细胞。修饰型线性化家蚕杆状病毒Bm-BacPAK6感染结果显示:病毒感染后转化BmN细胞和正常BmN细胞的BmSTAT基因表达水平分别提高了32.70%和14.63%,表明家蚕JAK/STAT途径对杆状病毒感染有应答。比较转化BmN细胞和正常BmN细胞对杆状病毒感染的抵抗性,发现病毒对转化BmN细胞的感染比例低于正常BmN细胞,二者之间的感染率相差8.28～18.02个百分点;在感染病毒的转化BmN细胞中,β-半乳糖苷酶基因的表达水平比病毒感染正常BmN细胞的略低,推测这与转化BmN细胞中的BmSTAT水平(JAK/STAT途径信号)高于正常BmN细胞有关。然而,在BmN细胞中,BmSTAT基因的过表达尚不足以明显提高细胞对病毒感染的抵抗力。研究结果为进一步探讨家蚕JAK/STAT途径在病毒感染应答方面的作用奠定了基础。     

视黄酸诱导基因-Ⅰ在家禽天然免疫应答中作用的研究进展

家禽的天然免疫应答在抵抗病毒感染的过程中起着关键性作用，视黄酸诱导基因-Ⅰ（retinoic acid inducible gene-Ⅰ，RIG-Ⅰ）作为细胞质内一类识别病毒双链RNA的模式识别受体，与天然免疫应答密切相关。它可通过RNA配体结合病原相关分子模式监测细胞质中的病毒RNA，此过程激活了RIG-Ⅰ及下游线粒体抗病毒信号蛋白（MAVS），最终导致干扰素调节因子（IRF3/7）和核因子κB （NF-κB）活化，诱导产生Ⅰ型干扰素等免疫细胞因子，进而使细胞做出相应的抗病毒天然免疫反应。但由于鸡体内缺乏RIG-Ⅰ基因，目前大多将鸭源或鹅源RIG-Ⅰ基因转染鸡成纤维母细胞（DF-1）研究RIG-Ⅰ基因在鸡感染禽类病毒时是否具有免疫功能。文章介绍了RIG-Ⅰ在家禽体内的表达及其介导的抗病毒天然免疫信号通路，并简述了RIG-Ⅰ在家禽体内抗病毒作用的研究概况，为抑制家禽病毒的感染和免疫系统研究，以及研制新型抗病毒疫苗或免疫佐剂等提供参考。     

Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype

Swine influenza virus (SIV) and Mycoplasma hyopneumoniae (Mhp) are widespread in farms and are major pathogens involved in the porcine respiratory disease complex (PRDC). The aim of this experiment was to compare the pathogenicity of European avian-like swine H1N1 and European human-like reassortant swine H1N2 viruses in na?ve pigs and in pigs previously infected with Mhp. Six groups of SPF pigs were inoculated intra-tracheally with either Mhp, or H1N1, or H1N2 or Mhp+H1N1 or Mhp+H1N2, both pathogens being inoculated at 21 days intervals in these two last groups. A mock-infected group was included. Although both SIV strains induced clinical signs when singly inoculated, results indicated that the H1N2 SIV was more pathogenic than the H1N1 virus, with an earlier shedding and a greater spread in lungs. Initial infection with Mhp before SIV inoculation increased flu clinical signs and pathogenesis (hyperthermia, loss of appetite, pneumonia lesions) due to the H1N1 virus but did not modify significantly outcomes of H1N2 infection. Thus, Mhp and SIV H1N1 appeared to act synergistically, whereas Mhp and SIV H1N2 would compete, as H1N2 infection led to the elimination of Mhp in lung diaphragmatic lobes. In conclusion, SIV would be a risk factor for the severity of respiratory disorders when associated with Mhp, depending on the viral subtype involved. This experimental model of coinfection with Mhp and avian-like swine H1N1 is a relevant tool for studying the pathogenesis of SIV-associated PRDC and testing intervention strategies for the control of the disease.     

甘肃省猪流感H5和H9抗体的血清学调查

用血凝（HA）和血凝抑制试验（HI）对甘肃省742份猪血清进行了猪流感H5和H9亚型抗体检测。在12个市州的猪群中检出SIH9亚型抗体,检出阳性34份,平均阳性率为4．58％。阳性率最高为9．09％,最低为0．91％。在2个市州的猪群中检出SIH5亚型抗体,检出阳性3份,平均阳性率为0．40％。阳性率最高为4．76％,最低为2．98％。检测患病猪（非流感病）血清66份,其中8份检测出SIH9抗体．抗体阳性率为12．12％。     

Isolation and genetic characterization of avian origin H9N2 influenza viruses from pigs in China

As pigs are susceptible to infection with both avian and human influenza A viruses, they have been proposed to be an intermediate host for the adaptation of avian influenza viruses to humans. In April 2006, a disease caused by highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) occurred in several pig farms and subsequently overwhelmed almost half of China with more than 2,000,000 cases of pig infection. Here we report a case in which four swine H9N2 influenza viruses were isolated from pigs infected by highly pathogenic PRRSVs in Guangxi province in China. All the eight gene segments of the four swine H9N2 viruses are highly homologous to A/Pigeon/Nanchang/2-0461/00 (H9N2) or A/Wild Duck/Nanchang/2-0480/00 (H9N2). Phylogenetic analyses of eight genes show that the swine H9N2 influenza viruses are of avian origin and may be the descendants of A/Duck/Hong Kong/Y280/97-like viruses. Molecular analysis of the HA gene indicates that our H9N2 isolates might have high-affinity binding to the alpha2,6-NeuAcGal receptor found in human cells. In conclusion, our finding provides further evidence about the interspecies transmission of avian influenza viruses to pigs and emphasizes the importance of reinforcing swine influenza virus (SIV) surveillance, especially after the emergence of highly pathogenic PRRSVs in pigs in China.     

How the bovine viral diarrhea virus outwits the immune system

The interaction of bovine viral diarrhea virus (BVD virus) with its host has several unique features, most notably the capacity to infect its host either transiently or persistently. The transient infection stimulates an antiviral immune reaction similar to that seen in other transient viral infections. In contrast, being associated with immunotolerance specific for the infecting BVD viral strain, the persistent infection differs fundamentally from other persistent infections like those caused by lentiviruses. Whereas the latter are characterized by complex viral evasion of the host's adaptive immune response by mechanisms such as antigenic drift and interference with presentation of T cell epitopes, BVD virus avoids the immune response altogether by inducing both humoral and cellular immune tolerance. This is made possible by invasion of the fetus at an early stage of development. In addition to adaptive immunity, BVD virus also manipulates key elements of the host's innate immune response. The non-cytopathic biotype of BVD virus, which is capable of persistently infecting its host, fails to induce type I interferon. In addition, persistently infected cells are resistant to the induction of apoptosis by double-stranded RNA and do not produce interferon when treated with this pathogen-associated molecular pattern (PAMP) that signals viral infection. Moreover, when treated with interferon, cells persistently infected with non-cytopathic BVD virus do not clear the virus. Surprisingly, however, despite this lack of effect on persistent infection, interferon readily induces an antiviral state in these cells, as shown by the protection against infection by unrelated viruses. Overall, BVD virus manipulates the host's interferon defense in a manner that optimises its chances of maintaining the persistent infection as well as decreasing the risks that heterologous viral infections may carry for the host. Thus, since not all potential host cells are infected in animals persistently infected with BVD virus, heterologous viruses replicating in cells uninfected with BVD virus will still trigger production of interferon. Interferon produced by such cells will curtail the replication of heterologous viruses only, be that in cells already infected with BVD virus, or in cells in which the heterologous virus may replicate alone. From an evolutionary viewpoint, this strategy clearly enhances the chances of transmission of BVD virus to new hosts, as it attenuates the negative effects that a global immunosuppression would have on the survival of persistently infected animals.     

PEDV编码蛋白拮抗宿主天然免疫的研究进展

猪流行性腹泻病毒(PEDV)属于冠状病毒家族成员,是近年来引起新生仔猪水样腹泻致死的主要病原之一,给养殖业带来了巨大威胁.病毒感染后,宿主细胞通过模式识别受体(PRRs)识别病原相关分子模式(PAMPs),促进I型干扰素等细胞因子的产生,进而抑制病毒的增殖.近年来的研究表明,PEDV能够通过其编码蛋白对宿主的抗病毒天然...     

Systemic and mucosal immune responses to H1N1 influenza virus infection in pigs

Influenza is a common respiratory disease in pigs, and since swine influenza viruses are zoonotic pathogens, they also pose human health risks. Pigs infected with influenza virus mount an effective immune response and are protected from subsequent challenge, whereas the currently available, inactivated-virus vaccine does not consistently confer complete protection to challenge. To develop and evaluate new vaccination strategies, it is imperative to fully understand the immune responses that are associated with protection following natural infection. Therefore, we have evaluated the phenotype and kinetics of immune responses to primary and re-challenge infection with H1N1 swine influenza virus in the pig. Through the use of isotype-specific antibody secreting cell ELISPOT assays, interferon-gamma ELISPOT assays and isotype-specific ELISAs on serum, nasal wash and bronchoalveolar lavage samples, we defined the humoral and cellular immune responses, both locally in the respiratory tract and systemically, to this viral infection. Virus-specific serum IgG, IgA, and HI titers all peaked 2-3 weeks after primary infection and did not substantially increase after re-challenge. The predominant virus-specific isotype in serum was IgG. Pigs responded with virus-specific IgG and IgA in both the upper (nasal washes) and lower (bronchoalveolar lavages) airways; IgA was the predominant isotype in both sites. Despite the fact that the pigs were completely protected from re-challenge, the antibody titers in the nasal washes increased. Results of the antibody-secreting cell ELISPOT assays demonstrated that the numbers of both IgG and IgA secreting cells in the nasal mucosa were dramatically higher than in any other tissue examined. In contrast, IFN-gamma secreting cells were predominantly localized to the spleen and tracheobronchial lymph nodes. These data will be helpful in the future development and evaluation of novel vaccines.     

一株欧洲类禽H1N1猪流感病毒的分离鉴定与反向遗传系统的建立

猪流感是猪常见的呼吸道传染病,临床以高热、呼吸困难、咳嗽和衰竭、迅速康复或死亡为特征。猪流感不仅给养猪业造成巨大损失,也严重威胁着人类健康。本研究从发病猪场中分离到1株H1N1亚型猪流感病毒,序列分析结果显示,分离毒株属于欧洲类禽猪流感H1N1亚型病毒。将分离毒株分别接种到MDCK与ST细胞,观察病毒的生长特性,结果显示分离的猪流感病毒在ST细胞中复制能力较强。采用RT-PCR技术分别扩增8个基因片段,克隆到流感病毒反向遗传系统,成功拯救出猪流感病毒毒株,测序结果显示拯救的猪流感病毒与亲本毒序列一致。本研究成功分离的猪流感病毒,以及建立的反向遗传技术为研究欧洲类禽猪流感病毒跨种传播的机制以及研发新型猪流感疫苗株奠定了基础。     

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.     

猪流感病毒LAMP检测方法的建立

本试验针对猪流感病毒（swine influenza virus,SIV）NP基因保守区域设计并合成6条引物,建立了SIV的环介导等温扩增（LAMP）检测方法,并进行了特异性、敏感性和重复性试验。结果显示,该方法可特异性检测H1N1、H1N2、H3N2、类禽H1N1亚型SIV及甲型H1N1流感病毒,但不能检测猪繁殖与呼吸综合征病毒、猪细小病毒、猪瘟病毒、猪伪狂犬病病毒、猪圆环病毒2型、日本乙型脑炎病毒;该方法的最低检测量为100拷贝/μL质粒DNA。结果表明建立的LAMP方法具有较高的敏感性和特异性,可用于SIV的快速检测。     

Response of calves to exposure with swine influenza virus

In calves inoculated with live swine influenza virus (SIV) A/sw/IL/75 (H1N1) intranasally, SIV was isolated for 7 days, and respiratory tract disease was observed. Antibody was detected in serum of inoculated calves from postinoculation day 9, and virus-neutralization antibody was demonstrated on postinoculation days 14 and 21. The primary response was low, but readily differentiated from the secondary response after calves were challenge exposed with homologous SIV. Pneumonic lesions were observed at necropsy, and histopathologic changes in airways and lungs were consistently found. Fluorescent staining revealed viral activity in epithelial cells of airways. The virus was transferred to healthy calves housed with inoculated calves.