Monoclonal antibodies (mAbs) are widely used in virus research and disease diagnosis.  The nucleoprotein (NP) of influenza A virus (IAV) plays important roles in multiple stages of the virus life cycle.  Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research.  In this study, two mAbs against the NP protein, 10E9 and 3F3, were generated with recombinant truncated NP proteins (NP-1 and NP-2) as immunogens.  The heavy-chain subclass of both 10E9 and 3F3 was determined to be IgG2α, and the light-chain type was κ.  Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10E9 and 3F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively.  We found that mAbs 10E9 and 3F3 reacted well with the NP protein of H1–H15 subtypes of IAV.  Both 10E9 and 3F3 can be used in immunoprecipitation assay, and 10E9 was also successfully applied in confocal microscopy.  Furthermore, we found that the 10E9-recognized ₈₄SAGKDP₈₉ epitope and 3F3-recognized ₃₂₀ENPAH₃₂₄ epitope were highly conserved in NP among all avian and human IAVs.  Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

在过去的几十年，国内外研究人员对动物和人感染高致病性禽流感进行了全球范围的广泛监测，然而有关北朝鲜的禽流感流行病学研究数据却很少。在2013和2014年，在北朝鲜的家禽中多次暴发高致病性禽流感，我们分离到H5N1亚型高致病性禽流感病毒，进化分析显示分离到的2株病毒的HA基因高度同源，均属于2.3.2.1c分支，我们分析认为引起2014年鸡场禽流感暴发的病毒可能是由2013年Tudan鸭场病毒经迁徙野鸟引入。本实验的数据提供了禽流感病毒可以由野鸟-水禽-陆禽进行传播的直接证据。因此，我们必需加强水禽禽流感的监测和控制，这对预防和控制高致病性禽流感至关重要。

【目的】H7亚型禽流感病毒(Avian influenza virus, AIV)和H5亚型一样，多数属于高致病性毒株，通常在禽类间传播，引起家禽出现严重的临床症状和高死亡率。从1996年至2012年，加拿大、意大利、墨西哥、荷兰、英国和美国出现了人感染H7亚型流感病毒(H7N2、H7N3和H7N7)的病例。2013年，中国报告了人感染H7N9流感病毒的事件，此后，该病毒持续在人和禽类中传播。因此，开展H7亚型禽流感病毒抗体检测工作成为一个重要问题。【方法】该研究用纯化的H7-AIV作为包被抗原，辣根过氧化物酶（Horseradish peroxidase, HRP）标记的单克隆抗体1H9（HRP-1H9）作为竞争抗体，采用棋盘滴定法建立了一种检测H7亚型AIV抗体的竞争性酶联免疫吸附实验(Competitive enzyme-linked immunosorbent assay, cELISA)。【结果】抗原包被的最佳浓度为5 μg mL^-1，血清稀释度为1/10，酶标抗体为1/3000。用ROC曲线分析法测定178份AIV阴性和368份AIV阳性血清（n=546）的cELISA临界值，当PI为40%时，cELISA的特异性和敏感性分别为99.15%和98.12%。该方法可检测H7Nx（N1--N4、N7--N9）禽流感病毒抗体，与H1 -- H6、H8 -- H15亚型禽流感病毒及鸡新城疫病毒(Newcastle disease virus, NDV)、传染性支气管炎病毒(Infectious bronchitis virus, IBV)和传染性法氏囊病病毒(Infectious bursal disease virus, IBDV)等常见禽类病毒无反应，具有良好的特异性。该方法与血凝素抑制试验的符合率为98.56%。重复性实验表明，批内和批间重复的变异系数均小于12%。【结论、创新性】综上所述，本研究利用H7亚型特异性酶标单克隆抗体作为竞争抗体，建立了cELISA抗体检测方法，其优势是酶标抗体与待检血清同时加入反应孔中进行竞争反应，与先加入待检血清，再加竞争抗体反应的常规方法比较，简化了操作步骤，缩短反应时间，为开展H7-AIV抗体的大量检测提供了一种简单、便捷的技术手段。

   In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China.  Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin (HA) cleavage site, leading to the emergence of a highly pathogenic virus.  The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza.  Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus.  One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established.  Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity.  The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs.  Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples.  Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.

Fungi capable of arsenic (As) accumulation and volatilization are hoped to tackle As-contaminated environment in the future. However, little data is available regarding their performances in field soils. In this study, the chlamydospores of Trichoderma asperellum SM-12F1 capable of As resistance, accumulation, and volatilization were inoculated into As-contaminated Chenzhou (CZ) and Shimen (SM) soils, and subsequently As volatilization and availability were assessed. The results indicated that T. asperellum SM-12F1 could reproduce well in As-contaminated soils. After cultivated for 42 days, the colony forming units (cfu) of T. asperellum SM-12F1 in CZ and SM soils reached 1010–1011 cfu g–1 fresh soil when inoculated at a rate of 5.0%. Inoculation with chlamydospores of T. asperellum SM-12F1 could significantly accelerate As volatilization from soils. The contents of volatilized As from CZ and SM soils after being inoculated with chlamydospores at a rate of 5.0% for 42 days were 2.0 and 0.6 μg kg–1, respectively, which were about 27.5 and 2.5 times higher than their corresponding controls of no inoculation (CZ, 0.1 μg kg–1; SM, 0.3 μg kg–1). Furthermore, the available As content in SM soils was decreased by 23.7%, and that in CZ soils increased by 3.3% compared with their corresponding controls. Further studies showed that soil pH values significantly decreased as a function of cultivation time or the inoculation level of chlamydospores. The pH values in CZ and SM soils after being inoculated with 5.0% of chlamydospores for 42 days were 6.04 and 6.02, respectively, which were lowered by 0.34 and 1.21 compared with their corresponding controls (CZ, 6.38; SM, 7.23). The changes in soil pH and As-binding fractions after inoculation might be responsible for the changes in As availability. These observations could shed light on the future remediation of As-contaminated soils using fungi.