Highly pathogenic avian influenza virus H5N1 infects alveolar macrophages without virus production or excessive TNF-alpha induction

Highly pathogenic avian influenza virus (HPAIV) of the subtype H5N1 causes severe, often fatal pneumonia in humans. The pathogenesis of HPAIV H5N1 infection is not completely understood, although the alveolar macrophage (AM) is thought to play an important role. HPAIV H5N1 infection of macrophages cultured from monocytes leads to high percentages of infection accompanied by virus production and an excessive pro-inflammatory immune response. However, macrophages cultured from monocytes are different from AM, both in phenotype and in response to seasonal influenza virus infection. Consequently, it remains unclear whether the results of studies with macrophages cultured from monocytes are valid for AM. Therefore we infected AM and for comparison macrophages cultured from monocytes with seasonal H3N2 virus, HPAIV H5N1 or pandemic H1N1 virus, and determined the percentage of cells infected, virus production and induction of TNF-alpha, a pro-inflammatory cytokine. In vitro HPAIV H5N1 infection of AM compared to that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to 25% vs up to 84%), lower virus production and lower TNF-alpha induction. In vitro infection of AM with H3N2 or H1N1 virus resulted in even lower percentages of infected cells (up to 7%) than with HPAIV H5N1, while virus production and TNF-alpha induction were comparable. In conclusion, this study reveals that macrophages cultured from monocytes are not a good model to study the interaction between AM and these influenza virus strains. Furthermore, the interaction between HPAIV H5N1 and AM could contribute to the pathogenicity of this virus in humans, due to the relative high percentage of infected cells rather than virus production or an excessive TNF-alpha induction.     

Immunofluorescent Cell Assay of Infectious Pancreatic Necrosis Virus

An immunofluorescent cell (IFC) assay technique was developed for the quantification of infectious pancreatic necrosis (IPN) virus of salmonid fishes. Cover slip cultures of rainbow trout gonad (RTG-2) cells were infected with diluted virus preparations. After incubation to permit antigen development, the cells were stained with antiviral fluorescent antibody, and the number of fluorescing (infected) cells was counted. Optimal conditions for the IFC assay procedure are: (i) the use of RTG-2 cells cultured for at least 3 days at 20 C; (ii) 1-h absorption of IPN virus to RTG-2 cells at 20 C or alternatively, 4 h at 4 C; (iii) staining the infected cell cultures at 10 to 12 h postinfection. A linear relationship between the relative concentration of virus in the inoculum and the number of fluorescent cells in the first cycle of infection was observed. The IFC assay method is more sensitive than the plaque method for the assay of IPN virus.     

Kinetics and mechanism of DNA repair. Preparation, purification and some properties of caged dideoxynucleoside triphosphates.

Caged dideoxyribosylthymine triphosphate, dideoxyadenosine triphosphate and arabinosylcytosine triphosphate were prepared in high yield by reaction with 1-(2-nitrophenyl)diazoethane at pH 4 and room temperature for 24 h. Synthesis of caged alpha-32P-labelled dideoxyadenosine triphosphate (approx. 5000 Ci/mmol) in 85% yield was achieved by a modification of the method used for the synthesis of the unlabelled compounds. ATP was shown to be an excellent buffer in the synthesis of alpha-32P-labelled material, and in caged form to be an effective carrier in h.p.l.c. purification. Preparative h.p.l.c. was used to achieve purification of unlabelled caged compounds to greater than 98% purity and 32P-labelled material to 97% purity. Photolysis of unlabelled and 32P-labelled caged compounds by using XeF-excimer laser irradiation at 351 nm was characterized by using difference spectrophotometry and h.p.l.c. analysis. The stability of caged dideoxyadenosine [a-32P]triphosphate in the presence of cultured mammalian cells was evaluated; the adenosine derivative is essentially stable for 1 h.     

Comparative large-scale propagation of retroviruses from Old World (Mason-Pfizer monkey virus) and New World (squirrel monkey virus) primates.

A cell culture method is described for the large-scale (50 to 150 1) production of Mason-Pfizer monkey virus and squirrel monkey virus, two primate retroviruses. Virus production was achieved with suspension cultures of chronically infected A204 human rhabdomyosarcoma cells harvested and clarified in the logarithmic stages of cell culture growth. Methods for the subsequent purification and concentration of virus material utilizing zonal centrifugation also are described. Applications of these methodologies resulted in products that afforded biochemical comparisons of these agents in a manner such that host cell-derived variations were minimized. These data indicated that high levels of production and efficient recovery and purification of virus material were achieved.     

The primary production of an infectious recombinant Herpes Simplex Virus vaccine

The production and extracellular release of a recombinant Herpes Simplex Virus (type 2) from monolayers of infected complementing Vero cells (CR2) are addressed. Growth and virus production conditions are identified that provide adequate virus titers with cell seeding densities and viral multiplicities of infection that could be reasonably handled in manufacturing. Harvesting by sonication of cell monolayers is shown to give the highest recovery of infectious virus (to 2.5 x 10(6) pfu/mL) but leads to process stream contamination by cellular proteins through the rupturing of cells (to 28 pg protein/pfu). By comparison, freeze-thaw cycles and osmotic rupture by hypotonic saline or glycerol shock procedures yield only low virus recovery (typically <10% of that by sonication), and are accompanied by yet higher levels of protein contamination (up to 30-fold higher pg protein/pfu). Addition of the polyanionic polymers, heparin or dextran sulphate to a harvest using either hypotonic saline, glycerol shock or isotonic phosphate buffered saline increased the yield of infectious virus in the supernatant. By contrast, addition of polycationic poly-L-lysine resulted in negligible increase in the supernatant virus titer. The highest virus titers (4.7 x 10(7) pfu/mL) were achieved following treatment of roller bottle cultured cells displaying a high cytopathic effect with heparin at 50 microg/mL for at least 3 h post harvest. This procedure also gave the lowest levels of protein contamination (<2 pg protein/pfu). The fivefold lower yield of infectious virus from cultures displaying a low cytopathic effect (<70% CPE) indicates the importance of cell physiological state at harvest.     

Comparative large-scale propagation of retroviruses from old world (mason-pfizer monkey virus) and new world (squirrel monkey virus) primates

Summary A cell culture method is described for the large-scale (50 to 150 1) production of Mason-Pfizer monkey virus and squirrel monkey virus, two primate retroviruses. Virus production was achieved with suspension cultures of chronically infected A204 human rhabdomyosarcoma cells harvested and clarified in the logarithmic stages of cell culture growth. Methods for the subsequent purification and concentration of virus material utilizing zonal centrifugation also are described. Applications of these methodologies resulted in products that afforded biochemical comparisons of these agents in a manner such that host cell-derived variations were minimized. These data indicated that high levels of production and efficient recovery and purification of virus material were achieved. This work was supported by Contract NO1-CO-25423 with the National Cancer Institute, National Institutes of Health.     

An HSV-1 Vector Expressing Tyrosine Hydroxylase Causes Production and Release of l-DOPA from Cultured Rat Striatal Cells

Abstract: In this report we demonstrate that a defective herpes simplex virus type one (HSV-1) vector can express enzymatically active tyrosine hydroxylase in cultured striatal cells that are thereby converted into l -DOPA-producing cells. A human tyrosine hydroxylase cDNA (form II) was inserted into an HSV-1 vector (pHSVth) and packaged into virus particles using an HSV-1 strain 17 mutant in the immediate early 3 gene (either ts K or D30EBA) as helper virus. Cultured fibroblasts were infected with pHSVth and 1 day later tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity were observed. The tyrosine hydroxylase enzyme activity directed the production of l -DOPA. pHSVth infection of striatal cells in dissociated cell culture resulted in expression of tyrosine hydroxylase RNA and tyrosine hydroxylase immunoreactivity. Release of l -DOPA and low levels of dopamine were observed from cells in pHSVth-infected striatal cultures. Expression of tyrosine hydroxylase and release of catecholamines were maintained for at least 1 week after infection.     

The affinity adsorptive recovery of an infectious herpes simplex virus vaccine

The chromatographic purification of a recombinant Herpes Simplex Virus (type 2) from salt- and heparin-released harvests of infected complementing Vero (CR2) cells is addressed. Functionalized matrices and process operating conditions are identified that provide adequate virus titres in eluates that are significantly reduced in CR2 cell protein and DNA and possess a low level of HSV-2 protein. Virus from diluted salt-released harvests (0.14 M NaCl) was not appreciably adsorbed onto either heparin-Sepharose or Cellufine-heparin matrices but was virtually completely adsorbed onto Cellufine-sulfate and heparin-HP matrices. Virus was recovered by either a linear salt gradient elution (0.14-2 M NaCl) or by a single-step elution with 1.5 M NaCl in phosphate buffer. Recoveries of infectious virus with step elution were 21% and 89%, respectively, for these matrices. Virus from undiluted salt-released harvest (0.8 M NaCl) was substantially adsorbed onto Cellufine-sulfate gel (44% adsorption) and completely adsorbed onto heparin-HP matrices. This virus was recovered with high yield by either gradient or step elution with phosphate-buffered saline. Finally, heparin-harvested virus was fed directly to these matrices and quantitatively adsorbed. The virus could be completely recovered from the heparin-HP matrix with 1.5 M NaCl buffer to provide a purified preparation containing only 0.05 pg protein/pfu and 1.2 x 10(-4) pg DNA/pfu.     

Requirement for Cell-to-Cell Contact in Epstein-Barr Virus Infection of Nasopharyngeal Carcinoma Cells and Keratinocytes

Two nasopharyngeal carcinoma (NPC) cell lines and one keratinocyte cell line could be infected with Epstein-Barr virus (EBV) by cocultivation with virus-producing cells but not by cell-free virus. Using porous culture inserts to manipulate the cell-to-cell contact, we demonstrated that contact between EBV donor B cells and EBV recipient epithelial cells was required for the infection. Cell-to-cell contact not only provided a CR2-independent route of infection but also enhanced CR2-mediated infection in a synergistic manner. Activity of two EBV promoters (Cp and Wp) and expression of EBNA2 were detected in the infected population. A small proportion of the infected cells spontaneously entered an EBV lytic state, which could be induced prominently by chemical treatment. This study provides information on how EBV may infect epithelial cells in vivo, such as at the onset of NPC development.     

Complement-mediated binding of naturally glycosylated and glycosylation-modified human immunodeficiency virus type 1 to human CR2 (CD21).

Particulate glycoproteins lacking sialic acid, such as desialylated enveloped viruses, readily activate complement through the alternative pathway. Human immunodeficiency virus type 1 (HIV-1) contains two heavily glycosylated and partially sialylated envelope glycoproteins: a surface gp120 and a transmembrane gp41. The abilities of naturally glycosylated HIV-1 and glycosylation-modified HIV-1 to interact with the complement system were examined with a biological assay which measured the binding of whole virus particles to cells expressing CR2 (CD21), the complement receptor found naturally in abundance on follicular dendritic cells and immature B cells. HIV-1 IIIB was synthesized in the presence or absence of the mannosidase II inhibitor, swainsonine, to give rise to high-mannose-type, nonsialylated, nonfucosylated carbohydrate moieties. The virus also was treated with neuraminidase or endo-beta-galactosidase to remove terminal sialic acids. An enzyme immunoassay specific for HIV-1 p24 core protein was used to quantitate the amount of virus bound to cell surfaces. Virus particles incubated with 1:3-diluted, fresh HIV-1-negative human serum as a source of complement readily bound to MT-2 (CD4+ CR2+) and Raji-3 (CD4- CR2+) cells but not to CEM (CD4+ CR2-) cells, suggesting that the virus bound to CR2 independently of CD4. Compared with heat-inactivated or C3-deficient sera, fresh complement increased binding by as much as 62 times for naturally glycosylated virus, and 5 times more than this for glycosylation-modified virus. Similar observations were made with freshly isolated, non-mitogen-stimulated peripheral blood mononuclear cells. Additional evidence that HIV-1 bound to CR2 independently of CD4 was provided by the fact that binding was blocked by monoclonal antibody OKB7 (anti-CR2) but not by OKT4a (anti-CD4). Also, the virus bound to transfected K562 cells (CD4-) which expressed recombinant human CR2 but did not bind to untransfected K562 cells. Results obtained with complement component-deficient sera indicated that binding required the alternative complement pathway. Raji-3 and transfected K562 cells could not be infected with HIV-1 in the presence of complement, suggesting that utilization of CR2 as a receptor in the absence of CD4 does not allow virus entry. The demonstration of CR2 as a receptor for HIV-1 in the presence of complement, together with the ability to enhance binding by desialylation, provides new insights into mechanisms of HIV-1-induced immunity and immunopathogenesis.     

Detection of Dengue Virus Antigens in Cultured Cells by Using Protein A-Gold-Silver Staining (pAgs) Method

A protein A-gold-silver (pAgs) staining was developed to detect dengue virus antigens in cultured cells. The method can be carried out in either newly-subcultured or monolayered cells. Dengue virus-inoculated C6/36 clone of Aedes albopictus cells and human endothelial cells appeared brown-yellowish color on the peripheral membrane of the infected cells. In many cases, the infected C6/36 cells appeared darker than that of the infected endothelial cells. The positive results from the inoculated C6/36 cells usually appeared as early as 2 days post-inoculation for types 1, 2, and 4 of dengue viruses and 3 days for the dengue 3 virus. The same batch of specimens detected by direct immunofluorescence antibody test (DFA) showed positive 4 days post-inoculation for the types 2, 3, and 4 of dengue viruses and 6 days for the dengue 1 virus. The result also showed that all pAgs-positive specimens were also DFA-positive, but not vice versa. It suggested that pAgs is not only sensitive but also specific for dengue virus detection from inoculated cultured cells.     

采用原位杂交及电镜检测CR2转染小鼠细胞的EB病毒感染

EB病毒不能感染小鼠是因为小鼠CR2受体构像与人的不同,通过对小鼠CR2受体进行定点空变,然后将野生型和突变型小鼠CR2／1（MCR2／1）及人CR2（hCR2）用基因转移技术导入小鼠鼻咽上皮细胞系（TMNE）进行表达,观察转染阳性细胞是否具有结合EB病毒的能力,EBER－1杂交结果显示,只有转染hCR2和空变型MCR2（mtMCR2)的TMNE细胞可以感染EB病毒,但是前感染EB病毒的阳性率比后高的高得多。电镜结果也进一步证实EB病毒可以感染这两种细胞,这为进一步研究EB病毒进入细胞的机制及建立EB病毒相关的鼻咽癌动物模型奠定了良好的基础。     

Defective Growth Correlates with Reduced Accumulation of a Viral DNA Replication Protein after Low-Multiplicity Infection by a Human Cytomegalovirus ie1 Mutant

To investigate the importance of the IE1 p72 regulatory protein during human cytomegalovirus replication, a recombinant virus unable to synthesize IE1 p72 was constructed. The Towne strain mutant CR208 lacked exon 4 of the major immediate-early gene and was isolated and complemented in an IE1-expressing immortalized human fibroblast line (ihfie1.3). Replication of CR208 in primary human fibroblasts was completed after an input multiplicity of 10 PFU/cell but was severely impaired at 0.1 PFU/cell. CR208 formed plaques with lower efficiency on primary fibroblasts than on ihfie1.3 cells, and the relationship between the CR208 inoculum size and the resulting number of undersized plaques was nonlinear, indicating that multiple particles of CR208 were required to initiate lytic replication in a single primary fibroblast. After infection of primary fibroblasts with CR208 at 5 PFU/cell, a normal pattern of viral antigens was detected, although IE1 p72 was absent. During lower-multiplicity infections, IE2 protein was consistently detected at similar levels in a similar proportion of CR208-infected cells relative to the case for a Towne infection, but many fewer CR208-infected cells contained the ppUL44 polymerase accessory protein when evaluated at 24 or 48 h after infection. Furthermore, fibroblasts infected with CR208 at a low multiplicity failed to form viral DNA replication compartments, despite having expressed IE2 p86. These low-multiplicity growth and expression defects were corrected in two rescued derivatives of CR208 able to synthesize IE1 p72. One rescued virus (CR249) carried a deletion removing the large intron between exons 1 and 2 of the ie1-ie2 locus, revealing that this intron was dispensable for growth in cell culture.     

Infection of cultured human airway epithelial cells by influenza A virus

The lack of an adequate in vitro model has hampered study of the cellular basis by which influenza A virus causes disease in the human airway. We report in vitro infection of human airway epithelial cells by influenza A virus. Fetal and adult human tracheal and bronchial epithelial cells cultured from explants and SV40 transformed adult human tracheal epithelial cells were exposed to a recently isolated strain of influenza A virus (H1N1) and a laboratory passaged strain (WSN) of influenza A virus at similar multiplicity of infection. All cultures derived from explants showed hemadsorption (approximately 30% of the cells) with the H1N1 virus. No hemadsorption was detected with the WSN virus. One of two transformed cell lines showed a 5-10% hemadsorption to cells after H1N1 exposure and none following exposure to WSN. Immunofluorescent staining for influenza A-specific antigens in virus-exposed, explant-derived cells indicated viral infection and replication in these cells. Hemagglutinating material in the growth medium of infected, explant-derived cell lines, increased as a function of time, indicating the production of virus proteins. Exposure of rhesus monkey kidney cells and new human tracheal epithelial cultures to supernatant from these cells resulted in hemadsorption, indicating the presence of infectious virus in the supernatant. Light microscopic examination of virally infected bronchial epithelial cells demonstrated that the common types of cytopathic changes were rarely seen while cell proliferation continued over time. The data indicate that influenza A virus can infect, replicate, and produce infectious virus in cultured human tracheal and bronchial epithelial cells.     

人集落刺激因子-1(CSF-1)对常见呼吸道病毒的抑制作用

本文观察集落刺激因子-1(CSF-1)及其诱生剂:内毒素(LPS)、胞壁二肽(MDP)和干扰素(IFN-α)对下列病毒所致细胞病变的抑制,包括不同型别腺病毒5株,单疱病毒Ⅰ型和Ⅱ型,流感病毒A_3型,鼻病毒,ECHO11型各1株,在人胚肺传代细胞株上病变抑制的结果如下:对HSV-1、HSV-2、腺病毒6、11、22型,流感A_3型,鼻病毒。(CSF-1)和IFN-α,一样有明显抑毒效果,LPS MDP联合使用对以上病毒有明显增强抑毒作用。CSF-1和IFN-α的抑毒作用能分别被CSF-1和IFN-α的抗体所解除。 CSF-1在人胚肺传代细胞和人胚皮肤肌肉传代细胞上对VSV的抑毒效果在人胚肺细胞中效果比人胚皮肤肌肉细胞更明显。LPS10ng/ml作用48小时比作用24小时效果更强。LPS MDP和IFN-α对二种细胞都有同样高效的抑毒作用。     

Inactivation or removal of the budded particles of a nuclear polyhedrosis virus of a silkworm, Bombyx mori L. (Lep., Bombycidae)

Effective methods to inactivate or remove budded particles of a nuclear polyhedrosis virus of Bombyx mori (BmNPV) from a cell-cultured media or from host haemolymph that is infected by this virus have been developed. Two types of suspensions containing BmNPV budded virus particles, TC-100 media that cultured BmN4 cells infected by this virus and haemolymph of B. mori larvae infected by this virus, were treated by 6% (w/v) polyethylene glycol (PEG), 0.01% (w/v) chitosan, 0.05% (v/v) linoleic acid (an emulsion), and/or diethylether. Treatment by linoleic acid followed by PEG-precipitation and treatment by diethylether followed by PEG-precipitation were so effective that these treatments suppressed the viral titre of BmNPV-infected larval haemolymph from an original titre (> 10⁹ TCID₅₀ units/ml) to below a detectable limit. These methods are suggested as being potentially useful in an insect factory system; that is, a protein production system utilizing a baculovirus vector and its insect host or cultured cells on a large scale.     

The elimination of ryegrass mosaic virus from Lolium multiflorum by meristem tip culture

Meristem tips were cultured from Lolium multiflorum plants infected with ryegrass mosaic virus (RMV). Meristem tips within the size range O'2-fi mm long were cultured on media with or without 2 ,4-D at 1 mg/1. The plants that regenerated in culture showed no symptoms over a 10 month period and no RMV particles were observed by electron microscopy. It was concluded that RMV had been eliminated. The method should prove useful in eliminating the virus from desirable genotypes used as parent material for seed production.     

The host range phenotype displayed by a Sindbis virus glycoprotein variant results from virion aggregation and retention on the surface of mosquito cells

The Sindbis virus variant NE2G216 is a PE2-containing host range mutant that is growth restricted in cultured mosquito cells (C6/36) due to inefficient release of virions from this cell type. The maturation defect of NE2G216 has been linked to the structures of N-linked oligosaccharides synthesized by arthropod cells. Analysis of C6/36 cells infected with NE2G216 by transmission electron microscopy revealed the presence of dense virus aggregates within cytoplasmic vacuoles and virus aggregates adhered to the cell surface. The virus aggregation phenotype of NE2G216 was reproduced in vertebrate cells (Pro-5) by the addition of 1-deoxymannojirimycin, an inhibitor of carbohydrate processing which limits the processing of N-linked oligosaccharides to structures that are structurally similar, albeit not identical, to those synthesized in C6/36 cells. We conclude that defective maturation of NE2G216 in mosquito cells is due to virion aggregation and retention on the cell surface and that this phenotype is directly linked to the carbohydrate-processing properties of these cells.     

Large scale production and downstream processing of a recombinant porcine parvovirus vaccine

Porcine parvovirus (PPV) virus-like particles (VLPs) constitute a potential vaccine for prevention of parvovirus-induced reproductive failure in gilts. Here we report the development of a large scale (25 l) production process for PPV-VLPs with baculovirus-infected insect cells. A low multiplicity of infection (MOI) strategy was efficiently applied avoiding the use of an extra baculovirus expansion step. The optimal harvest time was defined at 120 h post-infection at the MOI used, with the cell concentration at infection being 1.5x10(6) cells/ml. An efficient purification scheme using centrifugation, precipitation and ultrafiltration/diafiltration as stepwise unit operations was developed. The global yield of the downstream process was 68%. Baculovirus inactivation with Triton X-100 was successfully integrated into the purification scheme without an increase in the number of process stages. Immunogenicity of the PPV-VLPs tested in guinea pigs was similar to highly purified reference material produced from cells cultured in the presence of serum-containing medium. These results indicate the feasibility of industrial scale production of PPV-VLPs in the baculovirus system, safety of the product, and the potency of the product for vaccine application.