Npro of Bungowannah virus exhibits the same antagonistic function in the IFN induction pathway than that of other classical pestiviruses

Bungowannah virus is the most divergent atypical pestivirus that had been detected up to now, and does not fit into any of the four approved species: Bovine viral diarrhea virus type 1 (BVDV-1) and type 2 (BVDV-2), Classical swine fever virus (CSFV) and Border disease virus (BDV). However, the presence of N^pro and E^rns coding regions, which are unique to pestiviruses, provides clear evidence of a pestivirus. Nevertheless, the amino acid identity of Bungowannah virus N^pro and BVDV-1 N^pro (strain CP7) is only 51.5%. By using a BVDV-1 backbone, a novel chimeric construct was generated, in which the genomic region encoding the non-structural protein N^pro was replaced by that of Bungowannah virus (CP7_N^pro-Bungo). In vitro studies of CP7_N^pro-Bungo revealed autonomous replication with the same efficacy as the BVDV backbone CP7 and infectious high-titer virus could be collected. In order to compare the ability of interferon (IFN) suppression, two reporter gene assays, specific for type-I IFN, were carried out. In virus-infected cells, no significant difference in blocking of IFN expression between the parental virus CP7, Bungowannah virus and the chimeric construct CP7_N^pro-Bungo could be detected. In contrast, an N^pro deletion mutant showed an impaired replication in bovine cells and a marked type-I IFN response.Taken together, our findings reveal the compatibility of non-structural protein N^pro of atypical Bungowannah virus with a BVDV type 1 backbone and its characteristic feature as an inhibitor of type-I IFN induction with an inhibitor-activity comparable to other pestiviruses.     

草鱼IFNa和IFNd重组蛋白表达、纯化及单克隆抗体制备

为系统研究草鱼I型干扰素的合成、分泌和免疫功能,本研究在大肠杆菌中表达并提纯了草鱼IFNa（CiIFNa）和IFNd（CiIFNd）重组蛋白。将CiIFNa和CiIFNd成熟肽分别克隆到pET-21d或pEHISTEVb表达载体上,并转化到大肠杆菌中;IPTG诱导表达得到CiIFNa和CiIFNd成熟肽的包涵体,经过盐酸胍变性、蛋白复性和浓缩后,利用AKTA分子筛层析获得了纯度较高的重组蛋白。用重组蛋白免疫小鼠,通过PEG法诱导得到杂交瘤细胞;将稳定分泌抗体的阳性细胞株的细胞悬液注射入小鼠腹腔,制备腹水抗体并进行纯化。本研究纯化了草鱼CiIFNa和CiIFNd各2株抗体,并采用SDS-PAGE、ELISA、Western blot和免疫荧光法对其进行了较全面的鉴定。研究结果表明CiIFNa和CiIFNd单克隆抗体特异性好、效价高,能够特异识别在大肠杆菌和真核细胞中表达的重组蛋白,且不存在CiIFNa和CiIFNd分子间的交叉识别。本研究制备的单克隆抗体为深入研究草鱼干扰素的细胞来源和蛋白表达规律奠定基础。     

Oral Recombinant Feline Interferon-Omega as an alternative immune modulation therapy in FIV positive cats: Clinical and laboratory evaluation

Recombinant-Feline Interferon-Omega (rFeIFN-ω) is an immune-modulator licensed for use subcutaneously in Feline Immunodeficiency virus (FIV) therapy. Despite oral protocols have been suggested, little is known about such use in FIV-infected cats. This study aimed to evaluate the clinical improvement, laboratory findings, concurrent viral excretion and acute phase proteins (APPs) in naturally FIV-infected cats under oral rFeIFN-ω therapy (0.1 MU/cat rFeIFN-ω PO, SID, 90 days). 11 FIV-positive cats were treated with oral rFeIFN-ω (PO Group). Results were compared to previous data from 7 FIV-positive cats treated with the subcutaneous licensed protocol (SC Group). Initial clinical scores were similar in both groups. Independently of the protocol, rFeIFN-ω induced a significant clinical improvement of treated cats. Concurrent viral excretion and APP’s variation were not significant in the PO Group. Oral rFeIFN-ω can be an effective alternative therapy for FIV-infected cats, being also an option for treatment follow-up in cats submitted to the licensed protocol.     

HBV衣壳蛋白装配抑制剂的研究进展

乙型肝炎病毒(HBV)是一种常见的嗜肝性病原体,全球约有4亿人感染,带来严重的社会问题和经济负担。虽然目前已有预防和治疗HBV的药物,但由于耐药性等问题不能彻底清除病毒感染,新药开发依然迫在眉睫。介绍了HBV的病毒结构、复制和感染过程,对现有治疗药物的优缺点进行了总结。对以衣壳蛋白装配为靶点的新药研究进展进行综述,旨在为今后开发抗HBV的新药提供参考。     

鸡粒细胞巨噬细胞集落刺激因子-猪干扰素α1融合基因在大肠埃希菌中的可溶性表达及其生物学活性研究

为可溶性表达重组鸡粒细胞巨噬细胞集落刺激因子(ChGM-CSF)与猪干扰素α1(PoIFNα1)融合蛋白,并研究其生物学活性,本研究分别提取鸡、猪肝脏细胞总RNA,采用RT-PCR方法扩增ChGM-CSF和PoIFNα1基因,经linker连接上述两种基因后将其克隆于pET-32a原核表达载体,转化E.coliBL21(DE3)菌株进行诱导表达,SDS-PAGE和western blot检测融合蛋白表达产物。在ST细胞/VSV病毒测定系统以细胞病变抑制法滴定该融合蛋白的抗病毒活性;同时用MTT法检测其对鸡淋巴细胞增殖活性的促进作用。结果显示,PCR扩增并融合后的ChGM-CSF和PoIFNα1融合基因约为1000bp,构建重组表达载体后,诱导表达的rChGM-CSF-PoIFNα1融合蛋白分子量约55ku,主要存在于破碎菌体的上清中,表达量较高。Westernblot检测结果显示,该融合蛋白分别能够与ChGM-CSF多抗和PoIFNα单克隆抗体特异性结合,其抗病毒比活性约为1.1×10^6IU/mg,并且具有促进鸡淋巴细胞增殖的活性。本研究为rChGM-CSF-PoIFNα1重组融合蛋白的研制及其相关活性的测定提供实验依据。     

Association between IL-6-572C/G as well as IFNAR1-168G/C and hepatitis B virus infection in populations of Dai and Han ethnicities in Yunnan Province

ATM: To explore the association between IL-6-572C/G (rs1800796) as well as interferon alpha receptor 1 (IFNAR1)-168G/C (rs2257167) and prognosis after hepatitis B virus (HBV) infection in populations of Dai and Han ethnicities in Yunnan Province. METHODS: The blood samples were collected from Dai people and Han people, each nation including 100 healthy controls and 200 infected individuals (100 spontaneous recovery individuals and 100 chronic patients). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to identify the gene type. RESULTS: In Dai people, no significant difference was found between genetic polymorphism of -572C/G and prognosis after HBV infection. The differences of C and G alleles between spontaneous recovery group and chronic hepatitis B group, and healthy controls and HBV infection group were not statistically significant. Meanwhile, GG and CG genotypes were a vital protective factor for the person who developed into a chronic heptatitis B patient under the G allele dominance mode (GG+CG/CC) (P<0.05). In Han people, no statistically significance for IL-6-572C/G genotype and allele distribution in each group comparisons had been found, as well as the C allele recessive mode and C allele dominance mode. For the above 4 indicators, no statistically significant difference of IFNAR1-168C/G in Dai and Han people had been found.CONCLUSION: The GG+CG genotype of IL-6-572C/G may be a protective factor for the HBV-infected Dai people to develop into chronic hepatitis B patients. However, there is no significant association between the IFNAR1-168G/C polymorphism and prognosis after HBV infection in the 2 ethnicities.     

Induction of interferon production in mice by delipidated whole cells and fractions of the aerobic corynebacterium, Corynebacterium catarrhalis

The i.v. or i.p. injection of delipidated whole cells of an aerobic Corynebacterium, designated as C. catarrahalis, resulted in the production of a low titre of interferon in mice. Two fractions, a particulate fraction, designated as CBA p40, and a soluble fraction, designated as CBA LS, could be isolated from the delipidated whole cells and were found to lead to the formation of more or less high interferon levels according to the route of administration. Furthermore, the CBA p40 fraction, when injected i.p., elicited the production of a maximum titre of interferon at the 24th hour (viral-like interferon).     

Changes of IFN-γ, IL-6 and TNF-α levels in rats with severe pneumonia

AIM:To study the variety of cytokines in severe bacterial pneumonia of Sprague Dawleg (SD) rats. METHODS:A total of 60 SD rats were randomly divided into three groups: model Ⅰ group (n=24), model Ⅱ group (n=24), and control group (n=12). Rats in the model Ⅰ group and the model Ⅱ group were intratracheally instilled with suspension of klebsiella pneumoniae at different doses. Rats in the control group were intratracheally injected with 1 mL saline. On the 2nd, 4th and 6th day after intratracheal instillation, 1/3 rats in each group were killed to determine the concentration of IFN-γ, IL-6 and TNF-α in blood. RESULTS:The levels of IL-6 and TNF-α in model groups were higher than those in control group, while the level of IFN-γ was lower. The change of cytokines was more significant in the model Ⅱ group (severe pneumonia) than those in the model Ⅰ group. CONCLUSION:The cytokines we studied may play an important role in the pathogenesis of severe pneumonia. The change of cytokines is more significant in severe pneumonia than those in common pneumonia.     

The expression of IFN-γ and IL-4 on T lymphocytes that infiltrate in nasal polyps

AIM:To investigate the expression of Th1-typed cytokine IFN-γ and Th2-typed cytokine IL-4 on T lymphocytes that infiltrate in nasal polyps for searching the pathogenesis of nasal polyps. METHODS:Nasal polyps tissue samples and peripheral blood were obtained from 21 patients. Normal human inferior turbinate mucosa and peripheral blood were obtained as well. Flow cytometry was adopted to detect the expression of IFN-γ and IL-4 of T lymphocytes. RESULTS: Th cytokines were rarely detected in inferior turbinate from normal human. Nasal polyps tissue consisted of abundant T lymphocytes. The expression of IL-4 and IFN-γ increased in peripheral blood from patients compared with normal human (P<0.05). The expression of IL-4 increased but the expression of IFN-γ decreased in nasal polyps compared with that of peripheral blood from the same patient (P＜0.05). CONCLUSION:There were generous of T lymphocytes infiltrating in nasal polyps. There was abnormal immune status in the local nasal mucosa from the patients, and the predomination of Th cytokine secretion changed compared with peripheral blood from the same patients, which resulted in the change of microenvironment of nasal mucosa and possibly close related to the formation of nasal polyps.