Development of a capture ELISA to determine kinetics of soluble CD25 following in vitro and in vivo stimulation of duck peripheral blood monocytes

In humans and other mammals, the α-chain of interleukin-2 (IL-2) receptor (CD25) is induced and expressed on the cell surface after lymphocyte activation and is released from the membrane of activated cells as a smaller soluble form (sCD25). However, little is known about avian sCD25. In the present study, we developed an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) to detect serum sCD25 in ducks, and we used flow cytometry (FCM) to analyze the frequency of CD25(+) cells in the peripheral blood of ducks infected with H9N2 or H5N1 avian influenza virus (AIV) or serotype II Riemerella anatipestifer (RA). Using the AC-ELISA, duck sCD25 molecules were detected in the supernatant and lysates of concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC), and in the serum of ducks infected with H5N1 virus and RA. However, no sCD25 was detected in the serum of H9N2 AIV-infected ducks. FCM analysis revealed that CD25(+) cells were upregulated within the PBMC of RA-infected ducks throughout the experiment until death, while in the PBMC of H9N2- and H5N1 AIV-infected ducks, the frequency of CD25(+) cells increased in the early stage of infection and then returned to a lower level. Our findings confirm that the dynamics of sCD25 and CD25(+) cells are different in the peripheral blood of ducks infected with H9N2 virus, H5N1 virus, and RA.     

Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.     

Muscovy duck retinoic acid-induced gene I (MdRIG-I) functions in innate immunity against H9N2 avian influenza viruses (AIV) infections

Retinoic acid inducible gene I (RIG-I) is a cytosolic pattern recognition receptor that senses pathogen-associated molecular patterns (PAMPs). Muscovy duck (Cairina moschata) is a large duck different from other species of ducks, and is more susceptible to some microbial pathogens. In this study, the Muscovy duck RIG-I gene (MdRIG-I) was identified. Quantitative RT-PCR showed that MdRIG-I mRNA was widely expressed in different tissues, especially in those with mucosa. RIG-I null DF-1 cells transfected with DNA constructs encoding MdRIG-I or CARDs domain can activate IRF-3 and NF-κB to up-regulated activity of IFN-β promoter. The components of the signaling pathway downstream of RIG-I in mammalian cells including IRF-3, NF-κB, IFN-β and the IFN-stimulated genes Mx-1, PKR and MDA5 were significantly up-regulated in CARDs-overexpressing-DF-1 cells. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression in brain, spleen, lung and bursa were up-regulated in ducks challenged with H9N2 avian influenza virus (AIV), whose six internal genes were closely related to the H7N9 and H10N8 AIV. In vitro, DF-1 cells transfected with MdRIG-I plasmid can respond significantly to H9N2 AIV, evident through enhancement of IFN-β promoter activity and decreased virus titer. Altogether, these results indicated that MdRIG-I is a novel member of RLR gene family, engaging in the early stage of antiviral innate immunity.     

Astragalus polysaccharide enhances immunity and inhibits H9N2 avian influenza virus in vitro and in vivo

This study investigated the humoral immunization of Astragalus polysaccharide (APS) against H9N2 avian influenza virus (H9N2 AIV) infection in chickens.The effects of APS treatment on H9N2 infection was evaluated by an MTT [3(4, 5-dimethylthiazol-2-yl)-2, 3-diphenyl tetrazolium bromide] assay and analysis of MHC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to H9N2 AIV were enhanced in the first week after APS treatment. The results indicated that APS treatment reduces H9N2 AIV replication and promotes early humoral immune responses in young chickens.     

连翘水提液体外对禽流感病毒增殖及炎症因子表达的抑制效应

旨在评估连翘体外抗H5N1和H9N2禽流感病毒（avian influenza viruses,AIVs）增殖及其介导炎症的效果,本试验制备了连翘水提液,首先采用CCK-8法测定了连翘水提液对DF-1细胞的安全浓度,并通过3种处理方法（药液预处理病毒后感染细胞、先感染病毒后给药、先给药后感染病毒）来筛选连翘水提液的最佳给药方式;在最佳给药方式下,使用TCID₅₀法检测禽流感病毒H5N1和H9N2的增殖情况,并采用qRT-PCR检测了炎症相关趋化因子和细胞因子的表达变化。结果显示,连翘水提液对DF-1细胞的最高安全浓度为4 mg·mL^-1;先感染病毒后给药是最佳的给药方式;在最佳给药方式下,连翘水提液能显著降低H5N1和H9N2 AIVs在各个时间点的病毒滴度,并呈剂量依赖性关系;与对照组相比,H5N1 AIV给药组中CX3CL1、IL8L1、CCL5、SCYA4、IL1β、IL-6和TNF-α的表达显著降低,H9N2 AIV给药组中CX3CL1、IL8L1、CCL5、IFN-β、IL-6和TNF-α的表达也有类似的下降趋势。这表明连翘能够抑制H5N1和H9N2 AIVs在DF-1细胞中的增殖,降低炎症相关细胞因子的表达,有良好的抗炎和抗病毒活性。     

H7N2禽流感病毒CK/HB/1/02株NA全基因序列分析

采用RT-PCR扩增了国内H7N2禽流感病毒(AIV)CK/HB/1/02分离株的完整神经氨酸酶(NA)基因节段,测定了其核苷酸序列,并与GenBank中AIV NA基因序列进行了同源性比较和氨基酸编码分析,绘制了NA基因的系统发育进化树。结果表明,AIVCK/HB/1/02分离株NA基因的完整序列长度为1467bp,包括5′-末端和3′-末端的非编码区,其最大阅读框(ORF)编码469个氨基酸,第61~65位氨基酸序列为-NITGI-,不同于国内H9N2AIV的特殊遗传标记。该病毒NA基因的核苷酸序列与GenBank中人类流感病毒A/Leningrad/134/57(H2N2)的NA基因同源性最高,为93.3%;其次为香港的鸭源、人源、猪源H3N2病毒(89%~93%);而与我国北京、广东、香港和韩国的H9N2AIV以及美国的H7N2AIV的同源性较低(85%~88%)。在N2基因系统发育进化树中,CK/HB/1/02分离株处于欧亚分支内,与H2N2人流感病毒的亲缘关系较近;与北美H7N2AIV处在不同分支,遗传距离较远。     

Development of vaccine strains of H5 and H7 influenza viruses

To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokkaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/ Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 viruses as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9 N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.     

Molecular Characterization of a H9N2 Subtype Swine Influenza Virus

The objective of this study was to explore the epidemic situation and pathogenic characteristics of swine influenza virus (SIV) in Shandong Province. In the spring of 2019, 130 swine nasal swab samples were collected from a slaughterhouse in Tai'an city, Shandong Province for virus isolation and identification. The whole genome of isolated virus was sequenced and analyzed. Meanwhile, 1 527 swine serum samples were collected from swine farms in 8 regions of Shandong province and their anti-SIV antibody were detected by HI assay using standard avian H9N2 antigen. The results showed that a H9N2 subtype influenza virus strain was isolated and named as A/swine/Shandong/TA009/2019(H9N2). The homology analysis showed that the isolated virus had close genetic relationship with A/environment-air/Kunshan/NIOSH-BL20/2018(H9N2) and A/environment-air/Kunshan/NIOSH-BL25/2018(H9N2), and the nucleotide homology of the gene fragments were above 99.5%. Phylogenetic analysis results demonstrated that HA and NA genes of the isolated virus belong to the Y280-like lineage, PB2 and M genes belong to the G1-like lineage, and PB1, PA, NP and NS genes belong to the SH/F98-like lineage. The cleavage site in HA protein is “PSRSSR/GL”, which was in accordance with the molecular biological characteristics of low pathogenic avian influenza virus.The position 216 of HA protein is L, and it has the ability to bind human-derived sialic acid α 2,6-Gal. The results of HI showed that 9 among 1 527 serum samples were positive with a positive rate of 0.59%. The isolated virus was swine-derived H9N2 virus, and serological investigations revealed that H9N2 subtype virus infection was present in swine herds in Shandong Province. The results of this study suggest that continuous surveillance of the SIV epidemiological situation and its pathogenic characteristics should be strengthened.     

1株猪源H9N2亚型流感病毒的分子特征

旨在进一步了解山东省猪流感的流行情况及其病原特征,笔者于2019年春季,在山东省泰安某屠宰场采集130份猪鼻拭子,进行病毒分离鉴定;并对分离病毒进行全基因组测序和分子特征分析;用禽H9N2亚型标准抗原联合血凝抑制方法检测2018—2019年从山东省8个地区猪场采集的1 527份猪血清样品中的猪流感病毒抗体。结果显示：分离到1株H9N2亚型流感病毒,命名为A/swine/Shandong/TA009/2019（H9N2）。分离病毒与A/environment-air/Kunshan/NIOSH-BL20/2018（H9N2）和A/environment-air/Kunshan/NIOSH-BL25/2018（H9N2）遗传关系最近,其基因片段的核苷酸相似性均在99.5%以上。分离病毒的HA和NA基因属于Y280-like分支,PB2和M基因属于G1-like分支,PB1、PA、NP和NS基因属于SH/F98-like分支。分离病毒HA蛋白裂解位点处的氨基酸序列为“PSRSSR/GL”,符合低致病性禽流感病毒的分子生物学特性。HA蛋白的216位为L,具有结合人源唾液酸α 2,6-Gal的能力。血清学分析结果显示,9份血清中H9N2抗体呈阳性,其总阳性率为0.59%。综上：本研究分离到1株猪源H9N2亚型流感病毒,并在猪血清中检测到H9N2抗体,提示应加强对猪流感的流行情况及其病原特征的持续监测。     

H9N2亚型禽流感病毒中和单克隆抗体的制备与应用

为建立H9N2亚型禽流感病毒（Avian influenza virus,AIV）免疫层析快速检测技术,本研究以差速离心法纯化H9N2亚型AIV免疫BALB/c小鼠,将免疫小鼠脾细胞与骨髓瘤细胞SP2/0进行细胞融合和HAT选择性培养;以H9N2亚型AIV感染MDCK细胞建立异源免疫过氧化物酶单层细胞试验（IPMA）的单克隆抗体检测方法,通过对杂交瘤细胞的IPMA筛选和连续克隆化筛选鉴定抗H9N2亚型AIV中和性单克隆抗体;以胶体金标记HA单克隆抗体,配对HA单克隆抗体和羊抗小鼠IgG为检测线和质控线,制备H9N2亚型AIV快速检测试纸条,测定其特异性和敏感性。结果显示,获得了11株稳定分泌抗H9N2亚型AIV单克隆抗体的杂交瘤细胞,其单克隆抗体腹水IPMA效价在1.28×10^-4至2.56×10^-5之间。单克隆抗体3A2、5H6、6B8、7E10和9G12血凝抑制试验（HI）显示血凝抑制活性,其（HI）效价在6log2~9log2之间。单克隆抗体3A2、6B8和9G12在病毒中和试验中对H9N2亚型AIV有显著病毒中和活性,中和效价分别1∶6 400、1∶25 600和1∶25 600。Western blotting结果提示,该中和单克隆抗体识别HA蛋白线性抗原表位。利用配对单克隆抗体3A2和9G12研制的H9N2亚型AIV检测试纸条检测H9N2亚型AIV尿囊液的效价为9log2,灵敏度与经典血凝试验（HA）相当,与其他亚型AIV （H1、H3、H5、H7）,以及新城疫病毒和鸡传染性法氏囊病病毒等相关病毒均无交叉反应。本研究制备了具有病毒中和活性的抗H9N2亚型AIV单克隆抗体,并初步研制了H9N2亚型AIV检测试纸条,为H9N2亚型AIV新型疫苗研制和快速检测奠定良好的研究基础。     

小鼠细胞因子实时定量PCR检测方法的建立及应用

采用荧光SYBR Green I建立小鼠细胞因子mRNA实时定量PCR的检测方法;并利用建立的实时定量PCR的检测方法时感染H5N1禽流感病毒的BALB/c小鼠不同时间采集的肺脏组织中几种重要致炎细胞因子及趋化因子mRNA表达水平进行了检测.对HSN1禽流感病毒感染的BALB/c小鼠肺脏细胞因子的检测具有高度的特异性,检测的灵敏度为10~1～10~2拷贝数.H5N1禽流感病毒感染BALB/c小鼠后,小鼠肺脏中IL-1β、IL-6、TNFα、MIG、IP-10、RANTES、MIF和HMGB1细胞因子mRNA表达水平与时照组小鼠相比均有明显差异.建立的实时定量PCR能在基因转录水平敏感和特异地反应细胞因子的表达水平,该技术在基础和临床免疫研究中,具有良好的应用价值.     

鸭源H5N1亚型高致病性禽流感病毒的分离鉴定及其HA和NA基因的生物信息分析

分离到1株 H5N1亚型高致病性禽流感病毒, 经序列测定发现HA蛋白裂解位点上插入多个连续的碱性氨基酸（PQREIRRKKR*G）,从分子上证实是一株高致病性禽流感病毒。核酸序列比较分析结果表明,分离的流感病毒HA基因与A/duck/VietNam/Ncvd1/2002(H5N1)同源率最高,达到98.8%;NA基因与A/duck/VietNam/Ncvd1/2002(H5N1) 和A/chicken/Jiangsu/cz1/2002(H5N1)同源率最高,达到98.7%。氨基酸水平上,HA与A/duck/Viet Nam/Ncvd1/2002(H5N1)同源率最高,可达99.3%;NA与A/chicken/Jiangsu/cz1/2002(H5N1)同源率最高,达98.7%。HA与NA基因的潜在糖基化位点与作者所选参比毒株一致。通过遗传进化树分析结果表明,A/duck/VietNam/Ncvd1/2002(H5N1)可能是该毒株的来源株。     

太子参须提取物对鸭流感病毒体外试验初探

为尝试性探究太子参须提取物对鸭H9N2亚型流感病毒体外生长的抑制作用,本文基于TCID50试验检测鸭H9N2亚型流感病毒体外感染不同处理组的MDCK细胞.结果 表明,太子参须提取物高低剂量组(75％、25％)和黄芪多糖阳性对照组均对鸭H9N2亚型流感病毒体外生长产生抑制,且太子参须提取物高剂量组(75％)的抑制作用最优...     

A role for balance of interferon-gamma and interleukin-4 production in protective immunity against Neospora caninum infection

A suitable balance in the production of Th1/Th2-type cytokines has a crucial role in the control of microbial infections. We investigated cytokine production patterns and effects during Neospora caninum infection, based on two mouse models and an in vitro system. In the acute infection of N. caninum, BALB/c-background IFN-gamma-deficient mice that were sensitive to the N. caninum infection showed high levels of IL-10 production, whereas significant levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production were observed in resistant wild type mice. BALB/c mice vaccinated with recombinant vaccinia virus expressing N. caninum surface protein NcSRS2 resisted parasite spread throughout the body, low levels of IFN-gamma production and high levels of IL-4 production were observed compared to unvaccinated animals. The treatment of N. caninum-infected cells with IFN-gamma or IL-10 decreased the host-cell viability in an in vitro system using mouse macrophage J774A.1 cells. On the other hand, IL-4, but not IL-10 administration, increased the viability of N. caninum-infected and IFN-gamma-treated cells. In the light of the balance of Th1/Th2-type cytokine production, an IFN-gamma/IL-4 balance may have a crucial role for the control of cellular responses against the parasite invasion.     

Comparative molecular characterization,pathogenicity and seroprevalence of avian influenza virus H9N2 in commercial and backyard poultry flocks

This study was conducted to perform the comparative molecular characterization of avian influenza virus (AIV) H9N2, pathogenicity and seroprevalence in commercial and backyard poultry flocks. Fifty commercial poultry flocks were investigated between 2012 and 2015. Eighteen flocks (36%) out of 50 were positive HA. Seven (38.9%) out of 18 were positive by chromatographic strip test for AI common antigen. By Real-time RT-PCR, only two flocks were positive H9. The molecular characterization of two different AI-H9N2 viruses, one isolated from a broiler flock (A/chicken/Egypt/Mansoura-18/2013) and the other from a layer flock (A/chicken/Egypt/Mansoura-36/2015) was conducted on HA gene. Moreover, a higher seroprevalence, using the broiler strain as a known antigen, was shown in backyard chicken flocks 15/26 (57.7%) than duck flocks 9/74 (12.2%). Interestingly, the pathogenicity index (PI) of the H9N2 broiler strain in inoculated experimental chickens ranged from 1.2 (oculonasal route) to 1.9 (Intravenous route). The PI indicated a highly pathogenic effect, with high mortality (up to 100%) in the inoculated chickens correlated with the high mortality (80%) in the flock where the virus was isolated. The firstly recorded clinical signs, including cyanosis in the combs and wattles and subcutaneous haemorrhages in the leg shanks and lesions, as well as histopathology and immunohistochemistry, revealed a systemic infection of the high pathogenicity with the H9N2 virus. Conversely, the H9N2 layer strain showed a low pathogenicity. In conclusion, as a first report, the molecular analysis and pathogenicity of the tested strains confirmed the presence of a high pathogenicity AIV-H9N2 with systemic infections.     

An H9N2 influenza virus vaccine prepared from a non-pathogenic isolate from a migratory duck confers protective immunity in mice against challenge with an H9N2 virus isolated from a girl in Hong Kong

H9N2 influenza viruses circulate in wild birds and poultry in Eurasian countries, and have been isolated from pigs and humans in China. H9N2 viruses isolated from birds, pigs and humans have been classified into three sublineages based on antigenic and genetic features. Chicken antisera to H9N2 viruses of the Korean sublineage reacted with viruses of different sublineages by the hemagglutination-inhibition test. A test vaccine prepared from a non-pathogenic A/duck/Hokkaido/49/1998 (H9N2) strain of the Korean sublineage, obtained from our influenza virus library, induced immunity in mice to reduce the impact of disease caused by the challenge with A/Hong Kong/1073/1999 (H9N2), which is of a different sublineage. The present results indicate that an inactivated whole virus vaccine prepared from a non-pathogenic influenza virus from the library could be used as an emergency vaccine during the early stage of a pandemic caused by H9N2 infection.     

Efficient strategy for constructing duck enteritis virus-based live attenuated vaccine against homologous and heterologous H5N1 avian influenza virus and duck enteritis virus infection

Duck is susceptible to many pathogens, such as duck hepatitis virus, duck enteritis virus (DEV), duck tembusu virus, H5N1 highly pathogenic avian influenza virus (HPAIV) in particular. With the significant role of duck in the evolution of H5N1 HPAIV, control and eradication of H5N1 HPAIV in duck through vaccine immunization is considered an effective method in minimizing the threat of a pandemic outbreak. Consequently, a practical strategy to construct a vaccine against these pathogens should be determined. In this study, the DEV was examined as a candidate vaccine vector to deliver the hemagglutinin (HA) gene of H5N1, and its potential as a polyvalent vaccine was evaluated. A modified mini-F vector was inserted into the gB and UL26 gene junction of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The HA gene of A/duck/Hubei/xn/2007 (H5N1) was inserted into the C-KCE genome via the mating-assisted genetically integrated cloning (MAGIC) to generate the recombinant vector pBAC-C-KCE-HA. A bivalent vaccine C-KCE-HA was developed by eliminating the BAC backbone. Ducks immunized with C-KCE-HA induced both the cross-reactive antibodies and T cell response against H5. Moreover, C-KCE-HA-immunized ducks provided rapid and long-lasting protection against homologous and heterologous HPAIV H5N1 and DEV clinical signs, death, and primary viral replication. In conclusion, our BAC-C-KCE is a promising platform for developing a polyvalent live attenuated vaccine.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0174-3) contains supplementary material, which is available to authorized users.     

IBDV流行强毒HQ-b株与其细胞适应毒生物学特性比较及VP2、VP5基因序列分析

为了解IBDV流行强毒HQ-b株囊毒与其细胞适应毒间生物学特性差异及2毒株毒力变化与VP2、VP5基因变异的关系,对2毒株的细胞适应性、致病性等进行比较,同时对其VP2、VP5基因序列进行分析。结果表明,HQ-b株囊毒对CEF、CEK、CELi、DF-1和Vero均不适应,而细胞适应毒HQ株仅不能适应Vero细胞、且批内及批间毒价稳定。致病性结果显示HQ-b株对4周龄SPF鸡致死率高达80%,是真正的超强毒,而细胞适应毒致死率已降为0%。对VP2基因高变区研究表明,HQ-b株具备IBDV超强毒株的分子特征,即222A、256I、294I和299S;其细胞适应毒HQ株除222A→P、256I→V、294I→L和299S→N外,在VP2公认的毒力位点253(Q→H)、279(D→N)、284(A→T)位氨基酸也发生改变,导致细胞适应毒具备经典弱毒株的分子特征,即222P、256V、279N、284T、294L和299N。对VP5基因研究表明:流行强毒HQ-b株也具有IBDV超强毒株的分子特征;其细胞适应毒VP5基因有12个位点碱基突变并导致9处氨基酸变异,尤其是ORF区第2个碱基由"T"突变为"C"后,导致细胞适应毒VP5的N端丢失了4个氨基酸,这种突变与现有的疫苗毒完全一致。本研究提供了超强毒HQ-b培育、驯化后致病性和细胞适应性转变的分子机理,也丰富了IBDV分子流行病学的理论。