Detection of antibodies to the nonstructural protein (NS1) of influenza A virus allows distinction between vaccinated and infected horses.

Antibodies to the nonstructural protein (NS1) of A/equine/Miami/1/63 (H3N8) influenza virus were detected exclusively in the sera of mice experimentally infected with A/Aichi/2/68 (H3N2) and horses infected with A/equine/Kentucky/1/81 (H3N8) or A/equine/La Plata/1/93 (H3N8), but not in those of the animals immunized with the inactivated viruses, by enzyme-linked immunosorbent assay (ELISA) using a recombinant NS1 as antigen. The results indicate that the present method is useful for serological diagnosis to distinguish horses infected with equine H3 influenza viruses from those immunized with the inactivated vaccine.     

Detection of antibodies against porcine parvovirus nonstructural protein NS1 may distinguish between vaccinated and infected pigs

The humoral antibody response against the nonstructural protein NS1 and the structural protein VP2 of porcine parvovirus (PPV) was evaluated by immuno-peroxidase test (IPT) and enzyme linked immuno sorbent assay (ELISA) using recombinant PPV antigens. The coding sequence for NS1 and VP2 was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) genome resulting in two recombinant baculoviruses AcNPV-NS1 and AcNPV-VP2, respectively. Sf9 cells (Spodoptora frugidiperda) inoculated with AcNPV-NS1 producing recombinant nonstructural protein (rNS1) and AcNPV-VP2 producing recombinant virion protein (rVP2) were used in IPT and ELISA to analyse serum antibodies. Pigs vaccinated with an inactivated whole virus vaccine and experimentally infected pigs were studied. Significant titers against rVP2 were obtained in both vaccinated and infected pigs. Specific antibodies against rNS1 could only be detected in infected pigs and NS1 may in this way allow the specific detection of infected animals. Analysis of serum samples collected up to 18 days post infection (p.i.) from four pigs experimentally infected with PPV showed that antibodies against rNS1 and rVP2 could in all cases be detected on day 9 p.i. Two individual pigs were inoculated twice with PPV and the antibody response was followed 89 days after second inoculation. Serum antibodies against borth rVP2 and rNS1 could be detected for this period of time.     

禽流感病毒NS1蛋白与鸡LY6E蛋白间的相互作用

利用细菌双杂交系统,研究了禽流感病毒(Avian influenza virus,AIV)非结构蛋白(Nonstructurol1protein,NS1)和鸡淋巴细胞抗原复合体(LY6E)蛋白之间的相互作用。用RT-PCR方法克隆了AIV NS1基因和鸡LY6E基因的全长开放阅读框,将其克隆于细菌双杂交系统中的2个质粒中,成功构建了重组诱饵质粒pBT/NS1和重组目标质粒pTRG/LY6E。SDS-PAGE电泳表明,IPTG诱导后它们均得到正确表达。将重组质粒pBT/NS1和pTRG/LY6E共转化报告菌株,获得的阳性共转化子在含有3-AT和链霉素的组氨酸缺失的M9+筛选培养基上生长,表明AIV NS1蛋白和鸡LY6E蛋白之间存在相互作用,为AIV致病机制的研究提供了新的资料。     

The NS segment of H5N1 avian influenza viruses (AIV) enhances the virulence of an H7N1 AIV in chickens

Some outbreaks involving highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 were caused by avian-to-human transmissions. In nature, different influenza A viruses can reassort leading to new viruses with new characteristics. We decided to investigate the impact that the NS-segment of H5 HPAIV would have on viral pathogenicity of a classical avian H7 HPAIV in poultry, a natural host. We focussed this study based on our previous work that demonstrated that single reassortment of the NS-segment from an H5 HPAIV into an H7 HPAIV changes the ability of the virus to replicate in mammalian hosts. Our present data show that two different H7-viruses containing an NS-segment from H5–types (FPV NS GD or FPV NS VN) show an overall highly pathogenic phenotype compared with the wild type H7–virus (FPV), as characterized by higher viral shedding and earlier manifestation of clinical signs. Correlating with the latter, higher amounts of IFN-β mRNA were detected in the blood of NS-reassortant infected birds, 48 h post-infection (pi). Although lymphopenia was detected in chickens from all AIV-infected groups, also 48 h pi those animals challenged with NS-reassortant viruses showed an increase of peripheral monocyte/macrophage-like cells expressing high levels of IL-1β, as determined by flow cytometry. Taken together, these findings highlight the importance of the NS-segment in viral pathogenicity which is directly involved in triggering antiviral and pro-inflammatory cytokines found during HPAIV pathogenesis in chickens.     

禽流感病毒H5N1亚型NS1蛋白的真核表达及其在细胞中的分布

为构建禽流感病毒(AIV) H5N1亚型非结构蛋白NS1的真核表达载体,并鉴定其在哺乳动物细胞中的表达与分布,本研究采用RT-PCR技术,从甲型流感病毒的总RNA中扩增NS1全长基因,并将其克隆于pXJ40中,构建真核表达载体pXJ40-HA-NSl.将该重组质粒转染293T细胞,通过western blot方法鉴定表达的NS1蛋白;并以免疫荧光技术观察NS1在H1299细胞中的分布与定位.Western blot结果显示NS1基因编码蛋白获得表达,免疫荧光检测显示NS1蛋白主要存在于细胞核中.本研究为NS1蛋白功能和H5N1亚型AIV致病机制的研究奠定了基础.     

抗禽流感病毒NS1蛋白单克隆抗体的制备及其抗原表位的初步分析

为制备抗禽流感病毒(AIV)NS1蛋白的单克隆抗体(MAb)及鉴定其抗原表位鉴定,本研究以原核表达并纯化的NS1重组蛋白免疫BALB/c小鼠,采用淋巴细胞杂交瘤技术制备杂交瘤细胞,通过间接ELISA进行筛选,制备的D7和D9 2株能够稳定分泌抗NS1蛋白MAb的杂交瘤细胞,亚型鉴定均为IgG1型,轻链均为k链.Western blot鉴定表明,这2株MAbs均能够识别NS1重组蛋白.间接免疫荧光鉴定表明,这2株MAbs均能够识别真核表达的NS1蛋白.利用噬菌体展示技术得到D9对应的短肽WNLNTV,与NS1蛋白aa 182～aa 187基本匹配,提示182WNDNTV187为NS1蛋白的一个线性表位.该结果为进一步研究NS1蛋白的结构和功能以及建立诊断方法奠定了基础.     

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) in NS3 DNA vaccinated and naturally infected cattle

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) were investigated. cDNA encoding NS3 from type 1a BVDV was used to vaccinate five calves, another five calves remained unvaccinated. Three weeks after final vaccination animals were challenged intranasally with heterologous type 1a BVDV. Anti-NS3 antibodies were detected in only one animal post-vaccination. Partial protection from virus challenge was observed in the vaccinates. Virus was not isolated from nasal mucosa of two vaccinates, and virus clearance from nasal mucosa was faster in the vaccinates compared to the controls. While elevated rectal temperatures were evident in both groups 7 days post-challenge, the mean increase in the controls was twice that observed in the vaccinates. In conclusion, NS3 DNA vaccination induced humoral immunity in one calf, and prevented fever and virus establishment in the nasal mucosa in 2/5 calves, demonstrating the efficacy of NS3 vaccination, which may benefit future development of pestivirus and flavivirus vaccines.     

H9N2亚型禽流感病毒非结构蛋白基因NS1的表达及其单克隆抗体的研制

利用RT-PCR技术扩增获得了H9N2亚型禽流感病毒的NS1基因,ORF长度为654bp。成功构建了重组表达载体pET-NS1,将其转化BL21,并诱导表达出了相对分子质量大约为28 000的NS1融合蛋白。NS1融合蛋白经His trap Hp Kit柱子纯化,采用缓慢稀释和透析相结合的方法复性H9N2-NS1蛋白,获得纯度较高的NS1蛋白,以纯化的NS1免疫BALB/c小鼠,间接接ELISA方法筛选阳性克隆,对获得的抗H9N2-NS1单克隆抗体（McAbs）进行特异性分析;建立了2株稳定分泌抗H9N2-NS1McAbs的杂交瘤细胞系6B9和6C2;腹水的特异性鉴定结果表明,2株McAbs能特异性的识别H9N2-NS1,而与H5N2亚型AIV、H9N2亚型AIV、NDV、IBV、IBDV、ILTV、EDS76均不发生反应。Western blotting鉴定表明,所获得的单抗只与NSl蛋白条带反应。亚类显示,6B9为IgG1,6C2为IgG2b。结果表明,2株抗H9N2-NS1McAbs的制备对H9N2亚型禽流感病毒检测试剂盒的研制以及该病的防治有重要意义。另外,NSl蛋白单抗的成功研制为进一步研究NSl蛋白的结构、功能与细胞的相互作用机制及AI遗传变异规律奠定了一定的基础。     

Production of Brucella abortus-specific protein A-reactive antibodies (IgG2) in infected and vaccinated cattle

The IgG2 anti-Brucella antibody response of cattle to Brucella vaccination and infection was measured. Three groups of animals were studied; Group 1 contained 11 non-vaccinated cows, Group 2, 17 cows vaccinated with a low dose of Strain 19 vaccine and Group 3, 17 cows vaccinated with a high dose of Strain 19 vaccine. All animals were challenged at Week 33 with an infectious isolate of B. abortus (Strain 2308). Studies of the IgG2 antibodies response indicated an absolute correlation between anti-Brucella IgG2 levels and infection of the animal. All animals showing reciprocal titers of greater than or equal to 3000 (16 of 45 tested) were found to be positive for the challenge organism at slaughter. Animals with reciprocal IgG2 titers less than or equal to 1000 (29 of 45 tested) were found to be negative for the challenge organism at the time of slaughter. The predictive value of IgG2 antibody levels for infection held for animals in all three groups and consequently this suggests that monitoring of specific IgG2 anti-Brucella antibody levels may be of value in detection of Brucella-infected cattle.     

Maternal transfer of antibodies specific for avian influenza viruses in captive whooper swans (Cygnus cygnus)

The transfer of maternal antibodies to offspring can effectively protect against avian influenza virus (AIV) infection during early life in chickens and can prevent AIV spread by decreasing the overall percentage of the avian population susceptible to this pathogen. Herein, we evaluated maternal antibody transfer dynamics in whooper swans (Cygnus cygnus), which represent an important AIV host species. In total, 57 eggs from 19 nests were collected to study the relationship between egg yolk AIV-specific antibody concentrations and factors including egg size, laying order, maternal serum AIV antibody titer, and maternal body condition. Overall, we found that AIV-specific antibodies were present in the serum of 63.2 % of surveyed female swans and were transferred to 50.8 % of analyzed eggs. We found maternal AIV-specific antibody concentration and body weight to be positively correlated with egg yolk AIV antibody concentration, whereas egg laying order was negatively correlated with yolk antibody titer. Overall, these findings maternal transfer of AIV-specific antibodies may function as a key mechanism governing the dynamics of AIV infection in swan populations.     

Effects of avian infectious bronchitis virus (Arkansas strain) on vaccinated laying chickens

Twenty-four-week-old white leghorn layers were inoculated subcutaneously with a killed Newcastle-infectious bronchitis (Massachusetts type) virus (MIBV) vaccine. The birds were challenged 194 days later intraocularly with Arkansas strain of infectious bronchitis virus (AIBV). The challenged hens laid significantly (P less than 0.005) fewer eggs than the unchallenged layers, and the eggs laid by the challenged groups weighed significantly less (P less than 0.001) than those laid by the unchallenged groups. Further, the internal quality (Haugh units) and shell quality of eggs laid by the challenged hens were significantly (P less than 0.005) inferior to the quality of eggs from unchallenged hens, and the challenged hens laid more soft-shelled, misshapen, and small-sized eggs than the unchallenged hens. The Arkansas serum hemagglutination-inhibition (AIBV-HI) titers of challenged birds increased continuously through 29 days post-challenge. The MIBV hemagglutination-inhibition (MIBV-HI) titers of killed-MIBV-vaccinated birds decreased during the same period. The study indicates that killed MIBV vaccine offered no protection to birds exposed to AIBV. The same vaccine was quite effective against a homologous (MIBV) virus challenge.     

Novel purification method for recombinant 3AB1 nonstructural protein of foot-and-mouth disease virus for use in differentiation between infected and vaccinated animals.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for differentiation of animals infected with foot-and-mouth disease virus (FMDV) from vaccinated animals. The test was based on a highly pure and concentrated preparation of recombinant 3AB1 protein obtained by expression in a prokaryotic system, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electro elution. Experimental- and field-serum samples from naive, vaccinated, and infected cattle were tested for anti3AB1 antibody using the ELISA. A cutoff level was set at 35% of the maximum absorbance obtained with a positive control serum (FMDV-infected animal, 21 days postinfection [dpi]). This assay could detect antibodies from sera of animals experimentally infected by contact (n = 118) with a sensitivity of 97.5%. The specificity was 100%, based on negative test results obtained on 109 sera from naive animals. Remarkably, all sera from animals vaccinated either once (n = 102) or twice (n = 30) were negative. In addition, this 3AB1-ELISA could detect seroconversion at 7 dpi in animals inoculated intradermolingually. This assay constitutes an important tool for the rapid detection of FMDV outbreaks in a vaccinated population. In addition, it presents a reliable, economical, and simple method for testing large numbers of serum samples.     

Protection of chickens against highly pathogenic avian influenza virus (H5N2) by recombinant fowlpox viruses.

Two recombinant fowlpox viruses containing the avian influenza H5 hemaglutinin (HA) gene were evaluated for their ability to protect chickens against challenge with a highly pathogenic isolate of avian influenza virus (H5N2). Susceptible chickens were vaccinated with the parent fowlpox vaccine virus or recombinant viruses either by wing-web puncture or comb scarification. Following challenge 4 weeks later with highly pathogenic avian influenza virus, all birds vaccinated by the wing-web method were protected by both recombinants, while 50% and 70% mortality occurred in the two groups of birds vaccinated by comb scarification. Birds vaccinated with the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 90% and 100% mortality, respectively, following challenge. Hemagglutination-inhibition (HI) antibody levels were low, and agar-gel precipitin results were negative before challenge. Very high HI titers and positive precipitating antibody responses were observed in all survivors following challenge.     

Protein concentrations and immunosuppressive properties of serum in chickens experimentally infected with avian tumor viruses

Sera from chickens affected by Marek's disease or developing Rous sarcoma were investigated. There were changes in the protein fractions, and the amount of alpha and beta fractions was consistently increased. At the same time, immunosuppressive factors were found to inhibit the number of plaque-forming cells in the spleen of mice immunized with sheep red blood cells.     

Pathology of specific-pathogen-free chickens inoculated with H5N1 avian influenza viruses isolated in Japan in 2004

Specific-pathogen-free chickens inoculated with H5N1 highly pathogenic avian influenza (HPAI) viruses isolated in Japan in 2004 were investigated pathologically. The chickens inoculated intravenously with the viruses died within 26 hr after inoculation. Macroscopically, minimal necrosis of the tip of the comb, and hemorrhages of the palpebral conjunctiva, liver, cerebellum, and muscles were rarely observed. Histologically, dead chickens had minimal focal necrosis of hepatocytes with fibrinous thrombi in sinusoids, mild necrosis of splenic ellipsoids with fibrinous exudation, minimal necrosis of the brain, mild necrosis of epidermal cells of the comb with congestion of the lamina propria, and hemorrhages and edema of the lamina propria of the conjunctiva. Virus antigens were seen in the sinusoidal endothelial cells and hepatocytes in the liver, the capillary endothelial cells of the spleen, the capillary endothelial cells and cardiac myocytes in the heart, the capillary endothelial cells and necrotic nerve cells in the brain, the capillary endothelial cells in the lamina propria of the comb, the renal tubular epithelial cells, and the pancreatic acinar cells. The chickens inoculated by natural infectious routes died within 1-4 days after inoculation. Macroscopically, some chickens had hemorrhages in the conjunctiva, edematous swelling of the face and wattles, hydropericardium, hemorrhages of the proventriculus and bursa of Fabricius, increased secretion of tracheal mucus, and congestion and edema of lungs. Histologic lesions by natural infectious routes were similar to those by intravenous inoculation, except for the pancreatic necrosis. This study suggests H5N1 HPAI viruses isolated in Japan in 2004 cause pathologic conditions similar to natural cases.     

Consequence of Cryptosporidiosis on the immune response of vaccinated broiler chickens against Newcastle disease and/or avian influenza

The consequence of cryptosporidiosis on the immune response of vaccinated chickens against Newcastle disease and/or avian influenza was studied by using 240, 1 day old, male, white Hy-Line chicks and divided into 8 groups and subgroups. Each group or subgroup was consisting of 30 chicks (15?×?2 replicates). The first and second groups were kept as unvaccinated control, G1uninfected and G2 infected. G3, G4 and G5 contained 2 subgroups A&B (G3A, G3B, G4A, G4B, G5A and G5B). Chicks of subgroup A were vaccinated only while chicks of subgroup B were infected and vaccinated. These chicks were orally inoculated with 5?×?10⁵ oocysts of Cryptosporidium baileyi (C. baileyi) at 2 days of age. Chickens were vaccinated intraocular with live Newcastle disease (ND) vaccine (Hitchner on day 7th and LaSota on day 17th of chicken life) (G3) or vaccinated by subcutaneous route with Volvac®- H5N2- AI vaccine on day 10 of chicken life (G4). Last group (G5) was infected similarly and vaccinated with ND and AI vaccines with the same day, dose and route of vaccination for each one. Random blood samples were collected for 3 weeks post-vaccination for investigation of humoral immune response against Newcastle and/or avian influenza vaccines by the haemagglutination inhibition (HI) test. The results showed that H5N2 vaccine at day 10 of chicken life is effective in chickens indicated by the geometric mean of HI titer against AI virus. The findings of this study showed that the infection with Cryptosporidia in the broiler chicken has a depressive effect on the immune status of the birds vaccinated against ND and/or AI vaccination. Moreover, the obtained protection rates against challenge with virulent ND virus observed to be parallel to the results of HI- test. Also, by using 2 different antigens (one commercial and field prepared antigen) to avian influenza virus, lower Geometric mean (GM) HI titer were appeared in infected and vaccinated group than vaccinated group only. A study of the relative lymphoid organs weight such as bursa of Fabricius from the experimental chicks indicated that those organs were comparable between the groups infected-vaccinated and vaccinated only. Non significant variations in final live weight between uninfected control and infected groups were indicated. Also, H5N2-AI vaccination at 10 days old did not affect the final live weight. ND and/or AI Vaccination could not be a substitute to application of good hygienic measures and fecal examination of the birds especially for protozoal diseases such as cryptosporidiosis. It could be concluded that cryptosporidiosis could be one cause of ND and/or AI vaccination failure in poultry farms.     

鹅源H5N1亚型禽流感病毒感染雏鸡免疫器官细胞凋亡的研究

为研究鹅源H5N1亚型禽流感病毒(AIV)人工感染雏鸡免疫器官细胞凋亡的动态变化,本研究将50只1日龄SPF雏鸡随机分为两组。试验组雏鸡于7日龄,分别经鼻、眼、口同时感染105TCID50的鹅源H5N1亚型AIV,分别于感染后3d、4d、5d、7d和14d迫杀,采取胸腺、法氏囊和脾脏,应用TUNEL染色法和透射电镜技术观察其细胞凋亡的动态变化情况。结果显示,试验组雏鸡的胸腺和脾脏凋亡细胞数量在感染后3d～7d极显著高于对照组(p<0.01);法氏囊凋亡细胞数量在感染后3d～4d比对照组明显增加(p<0.05或p<0.01)。组织器官超微结构检测可见凋亡细胞核染色质固缩并凝结成块,聚集在核膜周围,呈新月状或环状;细胞质浓缩。表明鹅源H5N1AIV能够诱导感染雏鸡免疫器官发生细胞凋亡。     

禽流感分离株GD/1/96(H5N1)非结构蛋白结构建模

为预测H5N1亚型禽流感病毒(AIV)NS1蛋白全长结构,本研究以A/Goose/Guangdong/1/96(H5N1)株NS1蛋白氨基酸序列为目标序列,通过同源建模以及蛋白质结构叠合预测出NS1蛋白全长单体以及二聚体初始结构,采用约束分子动力学方法对初始结构进行优化,并利用立体化学、折叠可靠性、残基包装质量等评判方法对优化后的模型进行评估。结果表明所建立的NS1蛋白全长单体及二聚体结构模型具有很高的可信度,可用于基于结构的功能研究及虚拟筛选等工作。