Infection of Haematopoietic Stem Cells In Mice With Friend Virus Induced Erythroleukaemia

Abstract. Animals infected with conventional anaemia (FVA) or polycythemia-inducing (FVP) strains of the Friend virus develop lethal erythroleukaemia. A variant strain, RFV, induces an initially identical disease except that it spontaneously regresses in 50% of infected mice. to determine whether pluripotent stem cells as measured by spleen colony forming units (CFU-s) in leukaemic mice are productively infected with virus and whether their infection influences the outcome of the disease, we tested CFU-s from leukaemic mice for susceptibility to cytotoxicity by monospecific antiviral gp70 antiserum. Spleen CFU-s from progressively leukaemic (FVP, FVA and RFV) mice were productively infected with virus. CFU-s in RFV progressors became infected by 40 days post-virus inoculation. FVA and FVP progressors became infected between 15 and 21 days post virus. Infection of CFU-s was accompanied by an increase in the proportion of replicating (S phase) CFU-s in these populations. Spleen CFU-s from fully regressed RFV regressor mice were uninfected and remained so throughout the course of their disease. Bone marrow CFU-s in both regressors and progressors remained uninfected and were not induced to increased cell cycling.     

Ebola Virus Disease in Mice with Transplanted Human Hematopoietic Stem Cells

The development of treatments for Ebola virus disease (EVD) has been hampered by the lack of small-animal models that mimick human disease. Here we show that mice with transplanted human hematopoetic stem cells reproduce features typical of EVD. Infection with Ebola virus was associated with viremia, cell damage, liver steatosis, signs of hemorrhage, and high lethality. Our study provides a small-animal model with human components for the development of EVD therapies.     

Antibody Response in Mice Inoculated with DNA Expressing Foot-and-Mouth Disease Virus Capsid Proteins

Candidate foot-and-mouth disease (FMD) DNA vaccines designed to produce viral capsids lacking infectious viral nucleic acid were evaluated. Plasmid DNAs containing a portion of the FMDV genome coding for the capsid precursor protein (P1-2A) and wild-type or mutant viral proteinase 3C (plasmids P12X3C or P12X3C-mut, respectively) were constructed. Cell-free translation reactions programmed with pP12X3C (wild-type 3C) and pP12X3C-mut produced a capsid precursor, but only the reactions programmed with the plasmid encoding the functional proteinase resulted in P1-2A processing and capsid formation. Baby hamster kidney (BHK) cells also produced viral capsid proteins when transfected with these plasmids. Plasmid P12X3C was administered to mice by intramuscular, intradermal, and epithelial (gene gun) inoculations. Anti-FMD virus (FMDV) antibodies were detected by radioimmunoprecipitation (RIP) and plaque reduction neutralization assays only in sera of mice inoculated by using a gene gun. When pP12X3C and pP12X3C-mut were inoculated into mice by using a gene gun, both plasmids elicited an antibody response detectable by RIP but only pP12X3C elicited a neutralizing antibody response. These results suggest that capsid formation in situ is required for effective immunization. Expression and stimulation of an immune response was enhanced by addition of an intron sequence upstream of the coding region, while addition of the FMDV internal ribosome entry site or leader proteinase (L) coding region either had no effect or reduced the immune response.     

Suppression of Established Friend Virus Leukemia by Statolon: I. Demonstration of a Latent Infection in Clinically Normal Mice

A single inoculation of statolon into mice with established Friend virus (FV) leukemia can suppress the viral infection and produce a clinical remission lasting many months. Eventually, however, most of the mice develop characteristic FV leukemia. Persistence of FV activity in the spleens of mice during clinical remission can be demonstrated by cell transfer and histopathologic studies. Transfer to normal mice of a large number of spleen cells (10⁷) from mice in remission produces FV leukemia, and transfer of a small number of cells (10²) produces immunity to FV challenge. Histopathologic examination reveals clusters of abnormal FV leukemia-like cells directly beneath the capsules of the spleens of mice in clinical remission.     

A Novel Mucosal Vaccine Against Foot-and-Mouth Disease Virus Induces Protection in Mice and Swine

Epitopes of a foot-and-mouth disease virus (FMDV) capsid protein VP1 complex and a chimera of 6×His-tagged cholera toxin B subunit (hCTB) were expressed in Hansenula polymorpha and used together as a mucosal vaccine. Antibody and cytokine responses to VP1–hCTB vaccine and protection against FMDV were evaluated by ELISA and a virus challenge test in mice, respectively. VP1–hCTB directly enhanced the expression of interleukin-5 (IL-5) both in serum and supernatants of cultured spleen cells. After challenging suckling mice with 10⁵ FMDV (=50% lethal dosage per mouse) a greater protection was seen after intraperitoneal and intranasal vaccinations than after oral vaccination. In swine immunized with VP1–hCTB, immune responses were achieved after three administrations, and the vaccine protected swine (80%) when challenged with 10^6.5 FMDV (=50% infectious dosage per swine). These results demonstrated the possibility of using CTB as a mucosal adjuvant to elicit protective immune responses against FMDV. Houhui Song, Zhiliang Wang and Dongxia Zheng contributed equally to this work.     

Critical Role of Airway Macrophages in Modulating Disease Severity during Influenza Virus Infection of Mice

Airway macrophages provide a first line of host defense against a range of airborne pathogens, including influenza virus. In this study, we show that influenza viruses differ markedly in their abilities to infect murine macrophages in vitro and that infection of macrophages is nonproductive and no infectious virus is released. Virus strain BJx109 (H3N2) infected macrophages with high efficiency and was associated with mild disease following intranasal infection of mice. In contrast, virus strain PR8 (H1N1) was poor in its ability to infect macrophages and highly virulent for mice. Depletion of airway macrophages by clodronate-loaded liposomes led to the development of severe viral pneumonia in BJx109-infected mice but did not modulate disease severity in PR8-infected mice. The severe disease observed in macrophage-depleted mice infected with BJx109 was associated with exacerbated virus replication in the airways, leading to severe airway inflammation, pulmonary edema, and vascular leakage, indicative of lung injury. Thymic atrophy, lymphopenia, and dysregulated cytokine and chemokine production were additional systemic manifestations associated with severe disease. Thus, airway macrophages play a critical role in limiting lung injury and associated disease caused by BJx109. Furthermore, the inability of PR8 to infect airway macrophages may be a critical factor contributing to its virulence for mice.Airway macrophages (Mφ) (AM), located at the interphase between air and lung tissue, provide the first line of defense following inhalation of airborne pathogens, including influenza viruses. In addition to phagocytosis of virions and virus-infected cells (16, 24), infection of AM represents an early event in recognition of the virus by the innate immune system. Following intranasal (i.n.) infection of mice, influenza virus replicates productively in type II epithelial cells lining the respiratory tract. Murine Mφ are also susceptible to influenza virus infection and viral proteins are produced, but replication is abortive and infectious progeny are not released (52, 69), although recent studies suggest limited release from mouse Mφ exposed to highly pathogenic H1N1 and H5N1 viruses (44). Following exposure to influenza virus, Mφ synthesize and release proinflammatory cytokines and alpha/beta interferon (26, 27, 45, 55), which further limit viral replication and spread within the respiratory tract. Inflammatory responses in the airways must be tightly regulated to ensure rapid virus clearance while avoiding excessive or chronic inflammation that may damage the delicate tissue-air interface.Liposome-encapsulated dichloromethylene diphosphonate (clodronate or CL2-MDP) is taken up by phagocytic Mφ and accumulates in the cytosol, resulting in Mφ death and depletion (66). Administration of clodronate liposomes (CL-LIP) has been widely used to selectively deplete airway Mφ in mouse models without affecting circulating monocytes (for examples, see references 4, 6, 8, 36, 47, 64, and 71). While CL-LIP has been used predominantly in rodent models of infection, it is noteworthy that CL-LIP treatment of pigs, a natural host of influenza virus, resulted in enhanced morbidity and mortality following infection with a human H1N1 subtype virus (30). In murine studies, depletion of airway Mφ prior to influenza virus infection led to increased cytotoxic CD8⁺ T-cell responses in the lungs of virus-infected mice (71); however, treatment with CL-LIP 48 h after infection was associated with impaired CD8⁺ T-cell responses (41). Furthermore, CL-LIP treatment prior to intranasal infection of mice with a recombinant virus bearing the surface glycoproteins of the 1918 pandemic strain led to exacerbated disease and mortality (64). Treatment of mice with CL-LIP has been associated with enhanced virus replication (41, 64, 71); however, the mechanisms by which airway Mφ initiate and modulate inflammatory responses and disease after influenza virus infection have not been fully elucidated.We observed in a previous study that influenza A virus strains show marked differences in their abilities to infect murine Mφ in vitro and implicated the Mφ mannose receptor (MMR) (CD206), a C-type lectin (46), in infectious virus entry (48). Virus strain BJx109, a reassortant virus bearing the surface glycoproteins of A/Beijing/353/89 (H3N2) and internal components derived from A/PR/8/34 (H1N1) (PR8) bears a highly glycosylated hemagglutinin (HA) molecule and was shown to infect Mφ to high levels, while the HA of PR8 is poorly glycosylated and the virus infected Mφ very poorly. In the current study, we demonstrate that intranasal infection of mice with BJx109 leads to mild clinical disease, while infection with an equivalent dose of PR8 leads to severe disease and rapid death. Depletion of airway Mφ by intranasal administration of CL-LIP prior to and during infection with influenza viruses had little effect on the course of PR8 infection; however, BJx109 infection of Mφ-depleted animals led to severe disease and death. Severe disease was associated with enhanced virus replication, severe airway inflammation, and pulmonary edema and vascular leakage, indicative of lung injury. Together, these data demonstrate that airway Mφ play a critical role in moderating disease severity during BJx109 infection. Furthermore, they suggest that the ability of PR8 to evade infectious uptake by airway Mφ is likely to be an important factor contributing to its virulence for mice.     

卡介苗干预对哮喘小鼠肺组织IL-17A表达的影响及可能机制研究

目的:观察卡介苗干预对哮喘小鼠肺组织IL-17A的表达、气道炎症的影响,并探讨可能机制.方法:按随机数字表法,24只昆明小鼠分为正常对照(A组),模型组(B组),卡介苗(BCG)干预组(C组).C组小鼠每周一次皮内注射BCG 0.025 mg,连续3次.首次皮内注射4周后,第1、8、15天每只小鼠分别给予鸡卵清蛋白(OVA)与氢氧化铝混合腹腔注射,第22天给予1％ OVA溶液雾化吸入激发.激发后收集支气管肺泡灌洗液(BALF)行细胞总数及分类计数.肺组织HE染色行支气管粘膜下炎症评分.酶联免疫吸附试验法(ELISA)检测肺组织匀浆中的IL-17A和IFN-γ水平.结果:与正常对照组比较,BCG干预小鼠BALF中白细胞总数及中性粒细胞、嗜酸性粒细胞、淋巴细胞、巨噬细胞数均高于显著增高,低于模型组小鼠.BCG处理小鼠支气管粘膜下炎性细胞浸润程度较模型组小鼠明显降低(P＜0.01),比A组明显(P＜0.01).与对照组相比,模型组和BCG组小鼠肺组织匀浆中的IFN-γ显著相抵,在模型组小鼠更明显,IL-17A浓度增高,在模型组小鼠更明显(P＜0.05).结论:BCG干预可抑制气道炎症,可能通过上调Th1型免疫反应增加IFN-γ浓度、减少IL-17A在哮喘小鼠肺组织中表达,减少中性粒细胞的募集活化.     

浙江大蒜病毒的毒源鉴定

浙江余姚白蒜等S个品种均有病毒病,发病率100％,呈现黄条褪绿、花叶和畸形扭曲。人工接种蚕豆(Viciafaba)产生局部褐斑,墙生藜(Chenopodium murabe)上呈现褪绿点斑或隐症,豇豆(Vigna sinensis)上为坏死点斑或花叶。电镜检测病毒粒子为线形,长度为300—2250×11—12nm,其中550—650nm占29.6％、700—800hm占28％;超薄切片发现大量风轮状、环状、束状和涡轮状胞质内含体,未见片层状聚集体,叶绿体被破坏。致死温度在55℃以上。免疫电镜测试和洋葱黄矮病毒(OYDV)有血清学相关性,根据以上特征证明浙江大蒜病毒病是由两种病毒复合感染引起的,一种是大蒜黄条病毒(GYSV),属马铃薯Y病毒群(Potyviruses);另一种是大蒜隐潜病毒(GLV),属于石竹隐潜病毒群(Carlaviruses)。     

Some Surface-Active Agents and Their Virucidal Effect on Foot-and-Mouth Disease Virus

Selected cationic and anionic surface-active compounds were tested to determine their virucidal effect on the foot-and-mouth disease virus, type O, strain M11, propagated in primary calf kidney cells. The chemical inactivation of the virus was tested with 0.5, 1.0, 2.0, and 5.0% concentrations of the selected compounds. Virus controls with pH adjusted to cover the expected range of the mixtures of the chemicals and virus were also tested. The absence of virus from the mixtures of chemical and virus after reaction at 28 C for 2 hr was assayed by inoculating suckling mice with the mixtures. One cationic compound, alkyl methyl isoquinilinium chloride, showed considerable antiviral activity due largely to pH effect. The use of the surface-active agents investigated in this study, in the presence of organic material, would not be recommended as virucides.     

猪戊型肝炎核酸疫苗的构建及在BAL B/C小鼠免疫效应

将含有kozak序列及BamH I的上游引物和带有终止密码子及EcoRV酶切位点的下游引物,以猪HEVDQ1 ORF2为模板,进行PCR。将扩增片段和pcDNA3.1质粒以BamH I/EcoRV进行双酶切后进行连接。连接产物转化至大肠杆菌DH5α,经测序证明该序列正确,命名为pcDQ1。进行pcDQ1质粒提取,以Vero细胞为表达细胞进行转染,以间接免疫荧光试验进行验证。以100μg/次/只剂量的pcDQ1对BAL B/C小鼠进行免疫以获取单因子血清。共免疫3次,采集血清,进行ELISA效价测定。结果表明,该核酸疫苗可以免疫使小鼠产生抗体。     

Analysis of the Disease Potential of a Recombinant Retrovirus Containing Friend Murine Leukemia Virus Sequences and a Unique Long Terminal Repeat from Feline Leukemia Virus

We have molecularly cloned a feline leukemia virus (FeLV) (clone 33) from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV proviruses from other cats with AML, contains an unusual structure in its U3 region upstream of the enhancer (URE) consisting of three tandem direct repeats of 47 bp. To test the disease potential and specificity of this unique FeLV LTR, we replaced the U3 region of the LTR of the erythroleukemia-inducing Friend murine leukemia virus (F-MuLV) with that of FeLV clone 33. When the resulting virus, F33V, was injected into newborn mice, almost all of the mice eventually developed hematopoietic malignancies, with a significant percentage being in the myeloid lineage. This is in contrast to mice injected with an F-MuLV recombinant containing the U3 region of another FeLV that lacks repetitive URE sequences, none of which developed myeloid malignancies. Examination of tumor proviruses from F33V-infected mice failed to detect any changes in FeLV U3 sequences other than that in the URE. Like F-MuLV-infected mice, those infected with the F-MuLV/FeLV recombinants were able to generate and replicate mink cell focus-inducing viruses. Our studies are consistent with the idea that the presence of repetitive sequences upstream of the enhancer in the LTR of FeLV may favor the activation of this promoter in myeloid cells and contribute to the development of malignancies in this hematopoietic lineage.     

Malignant Transformation and Erythroid Differentiation by Polycythaemia-inducing Friend Virus

The polycythaemia-inducing Friend virus seems to transform erythropoietin-responsive stem cells, so that they are then able to differentiate into erythroblasts without erythropoietin.     

Transgenic Mice Expressing Human Immunodeficiency Virus Type 1 in Immune Cells Develop a Severe AIDS-Like Disease

We have constructed transgenic (Tg) mice expressing the entire human immunodeficiency virus type 1 (HIV-1) coding sequences in cells targeted by HIV-1 infection in humans. These Tg mice developed a severe AIDS-like disease leading to early death (<1 month). They developed muscle wasting, severe atrophy and fibrosis of lymphoid organs, tubulointerstitial nephritis, and lymphoid interstitial pneumonitis. In addition the expression of RANTES was increased in various tissues of these Tg mice relative to that in the normal controls. Disease appearance was correlated with the levels of transgene expression. The numerous pathologies observed in these mice are remarkably similar to those observed in human AIDS and, more specifically, in pediatric AIDS.