A RNA virus in cells from Culicoides variipennis

A virus was detected in cells (designated CuVa) cultured from one laboratory colony of the biting midge, Culicoides variipennis. By electron microscopy (30 nm), nonenveloped, icosahedral virions arranged separately and in crystalline matrix arrays were seen in the cytoplasm but not in the nucleus of CuVa cells. Separation by 10% polyacrylamide gel electrophoresis revealed multiple bands of viral-induced double-stranded RNA. Inoculation of this virus onto different cell lines and intracranially into suckling mice revealed no detectable pathology. Immunoperoxidase staining using polyclonal antibody determined that the virus is infectious to toad cells, bovine endothelial cells, bovine kidney cells, mosquito cells, and cells (designated KC) initiated from another laboratory colony of C. variipennis. KC cells infected with this virus were coinfected with bluetongue virus with no decrease in bluetongue virus titer.     

STRUCTURE AND DEVELOPMENT OF VIRUSES OBSERVED IN THE ELECTRON MICROSCOPE : IV. VIRUSES OF THE RI-APC GROUP

Representative viruses of the RI-APC group were observed with the electron microscope in thin sections of infected HeLa cells. The viral particles varied in density, were approximately 60 mµ in diameter and had a center to center spacing when close packed of about 65 mµ. Many of the less dense particles exhibited an internal body averaging 24 mµ in diameter. It was suggested that within the nucleus the virus differentiated from dense granular and reticular material and formed crystals. Disintegration of the crystals and disruption of the nuclear membrane with release of virus into the cytoplasm appeared to occur at any stage. No evidence to suggest development of the virus in the cytoplasm was obtained. It was possible to deduce the structure of the viral crystal from the electron micrographs. The viral particles are packed in a cubic body—centered lattice. Correlative histochemical observations in the light microscope which are now in progress revealed that the crystals and non-crystalline aggregates of virus were strongly Feulgen-positive.     

Vaccinia Virus Replication I. Requirement for the Host-Cell Nucleus

Using cytochalasin B-induced enucleation techniques, we examined the ability of vaccinia virus to replicate in the absence of the host-cell nucleus in several mammalian cell lines. It was found that virus-infected enucleated cells (cytoplasts) prepared from BSC-40, CVC, and L cells were incapable of producing infectious progeny virus. The nature of this apparent nuclear involvement was studied in detail in BSC-40 cells. Modulations designed to maximize cytoplast integrity and longevity, such as reduction of the growth temperature and initial multiplicity of infection, did not improve virus growth in cytoplasts. Sodium dodecyl sulfate-polyacrylamide gel analysis of the [(35)S]methionine pulse-labeled proteins synthesized in vaccinia virus-infected cytoplasts demonstrated that both early and late viral gene products were being expressed at high levels and with the proper temporal sequence. Vaccinia virus cytoplasmic DNA synthesis, as measured by [(3)H]thymidine incorporation, peaked at 3 h postinfection and was 70 to 90% of control levels in cytoplasts. However, in the cytoplasts this DNA was not converted to a DNase-resistant form late in infection, which was consistent with the failure to isolate physical particles from infected cytoplasts. Treatment of vaccinia virus-infected cells with 100 mug of rifampin/ml from 0 to 8 h to increase the pools of viral precursors, followed by subsequent removal of the drug, resulted in a threefold increase virus yield. This treatment had no effect on virus-infected cytoplasts. Finally, vaccinia virus morphogenesis was studied under an electron microscope in thin sections of virus-infected cells and cytoplasts which had been prepared at various times during a single-step growth cycle. It was apparent that, although early virus morphogenetic forms appeared, there was no subsequent DNA condensation or particle maturation in the cytoplasts. These results suggest that vaccinia virus requires some factor or function from the host-cell nucleus in order to mature properly and produce infectious progeny virus.     

狂犬病毒“北京株”在地鼠肾传代细胞中增殖的电镜观察

用透射电子显微镜观察术,对狂犬病固定毒“北京株”在地鼠肾传代细胞（ＢＨＫ２１）中增殖的形态学变化进行了研究。在受感染的细胞中有大量子弹状和长杆状的病毒颗粒。大多数病毒在扩张的内质网膜上以出芽的方式发生,凸向内质网腔内并逐渐发育成子弹状和杆关的成熟病毒。含病的内质,多周围常伴有颗粒或丝状均质区域,少数病毒在细胞膜上芽生。细胞间隙中亦可见病毒。还见病毒在细胞核膜内、外层上芽生,核周围间隙中许多病毒,有     

Electron Microscopic Study of the Development of Simian Virus 40 by Use of Ferritinlabeled Antibodies

An electron microscopic study of simian virus 40 has revealed a number of structural changes that are related to the development of the virus. The presence of viral antigens in both the nucleus and the cytoplasm has been demonstrated by means of ferritin-labeled antibodies. Although cytoplasmic virions are readily tagged, the lack of tagging of nuclear particles presents a perplexing problem. Presumably, the virus, after release from the nucleus, acquires a new antigenic reactivity in the cytoplasm.     

Ultrastructural characterization of an early, nonstructural polypeptide of herpes simplex virus type 1.

An immunoperoxidase procedure was employed to study the expression of a large-molecular-weight, virus-induced polypeptide (VP175; molecular weight, 175,000) at the light and electron microscopic levels in Vero cells infected with herpes simplex virus type 1 or with tsB2, a DNA-negative, temperature-sensitive mutant of herpes simplex virus type 1. In cells infected with herpes simplex virus type 1 and in cells infected with tsB2 at the permissive temperature (34 degrees C), VP175 was found within the nucleus. The protein was detected as early as 2 h postinfection and, by 3 h postinfection, was generally distributed in a marginated pattern contiguous with, and extending from, the inner lamella of the nuclear membrane. At 6 h postinfection, protein accumulations were dispersed throughout the nucleus, and, by 9 h postinfection, these accumulations tended to be localized in a marginated pattern near the nuclear membrane. It was also noted that, at 9 h postinfection, under permissive conditions, VP175 was not found in association with nucleocapsids or enveloped particles. In contrast, in cells infected with tsB2 at the nonpermissive temperature (39 degrees C) and harvested at 6 or 9 h postinfection, accumulations of VP175 were identified not only within the nucleus, but also within the cytoplasm in the form of annular or globular aggregates. These aggregates consisted of a granular matrix and were not bound by membranes.     

鸭肠炎病毒CHv强毒株超微结构研究

将鸭成纤维细胞培养的鸭肠炎病毒(DEV),经超声处理、高速冷冻差速离心后,采用酒石酸钾—甘油非线性密度梯度超速离心,收集病毒蛋白带,3%磷钨酸负染后观察病毒粒子形态。结果表明:病毒粒子主要集中在40%~50%酒石酸钾—甘油缓冲液交界层。电镜下,病毒粒子纯净,具有疱疹病毒典型形态结构,剖面六角,外观轮廓清楚。成熟病毒粒子直径约150~266nm,病毒囊膜、核衣壳和核心清晰可见;囊膜外层较内层着色略深,且可见尚未形成完整囊膜的柄状拖尾结构。多数病毒粒子以单核衣壳为主,一定数量的病毒具有双核衣壳,偶见三核衣壳,核衣壳直径为100~150nm,呈现致密圆形、半圆形或马蹄形等类型。在核衣壳外和囊膜之间可见明显的亮晕。核心DNA电子染色较深,集中分布,直径40~65nm。本文获得的清晰DEV负染超微结构照片,为该病毒结构生物学的研究提供了重要依据。     

Immuno-Electron Microscopy of the Morphogenesis of Mumps Virus

The fine structure of mumps virus-infected chick embryo fibroblastic cells was examined sequentially after viral inoculation. Intracytoplasmic nucleoprotein strands, similar to those described for parainfluenza viruses, were detectable in small aggregates between 36 and 48 hr. The peripheral strands of this viral component lie beneath and along an antigenically altered bulging portion of the cell membrane. The outermost strands are consistently parallel to the differentiated segment of the plasma membrane, which is invariably associated with surface projections. As has been found with other myxoviruses, mumps virus replicates by budding from the cell surface. The virus particle, roughly spherical in shape, has a size ranging from 1,000 to 8,000 A. Filamentous forms are rarely observed in the present culture system. Ferritin-conjugated antibody specifically labels the cytoplasmic nucleoprotein, the modified cell membrane, and the virus particle. Intranuclear inclusions of low electron density and morphologically different from those described in measles virus-infected HeLa and amnion cells were observed in the nucleus of several infected cells. Immuno-electron microscopic observations suggest that the nucleoprotein synthesis rate exceeds that of cell membrane differentiation into viral envelope. This difference results in the accumulation of viral nucleoprotein in large intracytoplasmic masses which can be demonstrated by electron microscopy.     

Labile Coat: Reason for Noninfectious Cell-free Varicella-Zoster Virus in Culture

Experiments designed to determine why cell-free varicella-zoster virus replicated in cell culture is noninfectious were performed. Electron micrographs in which varicella-zoster virus (a herpesvirus) was compared to herpes simplex virus in primary human amnion cell cultures showed that the viruses were morphologically indistinguishable inside the nucleus. However, extranuclear varicella-zoster viruses were distinguished from herpes simplex virus by the presence of pleomorphism, incomplete coats, and a resultant loss of central dense cores. This result indicates that varicella-zoster virus possesses a labile coat which is degraded outside the nucleus. It is suggested that the labile coat is a principal reason for the lack of cell-free infectious virus in this system.     

Epstein-Barr virus BNRF1 protein allows efficient transfer from the endosomal compartment to the nucleus of primary B lymphocytes

Epstein-Barr virus (EBV) is a tumor virus with marked B lymphotropism. After crossing the B-cell membrane, the virus enters cytoplasmic vesicles, where decapsidation takes place to allow transfer of the viral DNA to the cell nucleus. BNRF1 has been characterized as the EBV major tegument protein, but its precise function is unknown. We have constructed a viral mutant that lacks the BNRF1 gene and report here its in vitro phenotype. A recombinant virus devoid of BNRF1 (DeltaBNRF1) showed efficient DNA replication and production of mature viral particles. B cells infected with the DeltaBNRF1 mutant presented viral lytic antigens as efficiently as B cells infected with wild-type or BNRF1 trans-complemented DeltaBNRF1 viruses. Antigen presentation in B cells infected with either wild-type (EBV-wt) or DeltaBNRF1 virus was blocked by leupeptin addition, showing that both viruses reach the endosome/lysosome compartment. These data were confirmed by direct observation of the mutant virus in endosomes of infected B cells by electron microscopy. However, we observed a 20-fold reduction in the number of B cells expressing the nuclear protein EBNA2 after infection with a DeltaBNRF1 virus compared to wild-type infection. Likewise, DeltaBNRF1 viruses transformed primary B cells much less efficiently than EBV-wt or BNRF1 trans-complemented viruses. We conclude from these findings that BNRF1 plays an important role in viral transport from the endosomes to the nucleus.     

Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements

Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.     

AN ELECTRON MICROSCOPE STUDY OF TOBACCO MOSAIC VIRUS LESIONS IN NICOTIANA GLUTINOSA L

Ultra-thin sections of Nicotiana glutinosa L. leaves inoculated with a concentrated solution of tobacco mosaic virus were made at short intervals from 0 to 78 hours after inoculation. Eight hours after inoculation, the size of starch grains increased. This was followed by rupture of cytoplasmic and chloroplast membranes. At about 24 hours there was a great increase in number of mitochondria, which persisted until about 60 hours, when some became electron opaque while others appeared to disintegrate. Finally, the cell contents were compressed into one area of the cell, where they became electron opaque. This was accompanied by collapse of the rest of the cell and tearing away of the cell walls from adjacent cells. The nucleus remained stable and intact for as long as observations could be made. No identifiable virus particles were seen.     

Exploitation of microtubule cytoskeleton and dynein during parvoviral traffic toward the nucleus

Canine parvovirus (CPV), a model virus for the study of parvoviral entry, enters host cells by receptor-mediated endocytosis, escapes from endosomal vesicles to the cytosol, and then replicates in the nucleus. We examined the role of the microtubule (MT)-mediated cytoplasmic trafficking of viral particles toward the nucleus. Immunofluorescence and immunoelectron microscopy showed that capsids were transported through the cytoplasm into the nucleus after cytoplasmic microinjection but that in the presence of MT-depolymerizing agents, viral capsids were unable to reach the nucleus. The nuclear accumulation of capsids was also reduced by microinjection of an anti-dynein antibody. Moreover, electron microscopy and light microscopy experiments demonstrated that viral capsids associate with tubulin and dynein in vitro. Coprecipitation studies indicated that viral capsids interact with dynein. When the cytoplasmic transport process was studied in living cells by microinjecting fluorescently labeled capsids into the cytoplasm of cells containing fluorescent tubulin, capsids were found in close contact with MTs. These results suggest that intact MTs and the motor protein dynein are required for the cytoplasmic transport of CPV capsids and contribute to the accumulation of the capsid in the nucleus.     

Electron microscopic studies of circular DNA in mouse embryo fibroblasts infected by Rauscher leukemia virus.

Using electron microscopy, a closed circular form of DNA (4.3 mum in contour length) was detected in the nucleus of mouse embryo fibroblasts 2.5 h after infection by Rauscher murine leukemia virus. These circles were distinguishable from mitochondrial DNA by various criteria, including size, absence of secondary features, and resistance to EcoRI endonuclease.     

Effect of cytochalasin D on cell morphology and AcMNPV replication in a Spodoptera frugiperda cell line

Cytochalasin D (CD) is a specific inhibitor of actin microfilament elongation and has been used to identify actin-dependent cellular processes. In this study we observed the effects of this inhibitor on Autographa californica M nuclear polyhedrosis virus infected and uninfected IPLB-SF-21 cells by electron microscopy. The cytochalasin D-induced morphological effects detected in uninfected cells included lobulate nuclei, double nuclei, long retraction processes, increased zeiosis, more frequent plasma membrane indentations, increased vacuolation, more numerous coated pits and vesicles, filamentous masses in the cytoplasm, and decreased surface microvilli. Observation of infected cells treated with CD revealed that viral morphogenesis was severely affected. Few normal-appearing nucleocapsids were seen in the nucleus, and none were detected in the cytoplasm. Instead, long capsid-like tubular structures appeared juxtaposed to the inner nuclear membrane. Very infrequently sections of these structures contained electron dense material. The center of the nucleus contained electron-dense, spidery-like structures, presumably viral DNA. Normal virus was not observed to bud from the plasma membrane but electron-lucent, coreless-particles were. By 50 hr postinfection occasional polyhedra appeared, but these contained few or no enveloped virions. The intranuclear fibrous masses normally associated with infection were significantly reduced. These observations suggest that viral morphogenesis, especially nucleocapsid assembly, is an actin-dependent process.     

Replication of an entomopoxvirus in two lepidopteran cell lines

Pseudaletia separata entomopoxvirus replicated in two lepidopteran cell lines, SIE-MSH-805-F and BM-N. Microscopic examination, and the virus passage tests, of infected cultures indicated that the virus replicated more readily in the former cell line. Virus release by exocytosis occurred in both cell lines. A sequence of virus morphogenesis in the cultured cells was described, based on electron microscopic observations of thin sections. The nucleus of infected cells contained spherical inclusions, and the cytoplasm contained virions, immature virus forms, spheroids, and spindles. A portion of the virions in the cytoplasm was occluded within spheroids, which were often associated with crystallogenic matrix. Virions acquired a coat prior to their occlusion.     

Analysis of capsid formation of human polyomavirus JC (Tokyo-1 strain) by a eukaryotic expression system: splicing of late RNAs, translation and nuclear transport of major capsid protein VP1, and capsid assembly

Human polyomavirus JC (JCV) can encode the three capsid proteins VP1, VP2, and VP3, downstream of the agnoprotein in the late region. JCV virions are identified in the nucleus of infected cells. In this study, we have elucidated unique features of JCV capsid formation by using a eukaryotic expression system. Structures of JCV polycistronic late RNAs (M1 to M4 and possibly M5 and M6) generated by alternative splicing were determined. VP1 would be synthesized from M2 RNA, and VP2 and VP3 would be synthesized from M1 RNA. The presence of the open reading frame of the agnoprotein or the leader sequence (nucleotides 275 to 409) can decrease the expression level of VP1. VP1 was efficiently transported to the nucleus in the presence of VP2 and VP3 but distributed both in the cytoplasm and in the nucleus in their absence. Mutation analysis indicated that inefficiency in nuclear transport of VP1 is due to the unique structure in the N-terminal sequence, KRKGERK. Within the nucleus, VP1 was localized discretely and identified as speckles in the presence of VP2 and VP3 but distributed diffusely in their absence. These results suggest that VP1 was efficiently transported to the nucleus and localized in the discrete subnuclear regions, possibly with VP2 and VP3. By electron microscopy, recombinant virus particles were identified in the nucleus, and their intranuclear distribution was consistent with distribution of speckles. This system provides a useful model with which to understand JCV capsid formation and the structures and functions of the JCV capsid proteins.     

Observations of Measles Virus Infection of Cultured Human Cells : I. A Study of Development and Spread of Virus Antigen by Means of Immunofluorescence

The development of measles virus in cultures of both primary human amnion cells and H.Ep.-2 cells has been followed by means of the indirect fluorescent antibody technic and concurrent light and electron microscope observations. The immunofluorescence studies revealed that there is a latent period for development of demonstrable measles virus antigen. In amnion cells the latent period lasted for at least 3 days. In contrast, virus antigen could be detected in H.Ep.-2 cells as early as 12 hours following inoculation. In each cell system virus antigen was seen in either nucleus or cytoplasm of infected cells, or both. Early localization tended to be perinuclear. Intranuclear fluorescence was generally less bright and less widespread than cytoplasmic fluorescence. Giant cells and long cytoplasmic spindle-shaped processes appeared regularly in infected cultures. Infectious virus was liberated into the nutrient fluid but when extracellular virus was inhibited by antibody, spread of infection from cell to cell in the monolayer still continued. Results obtained in concurrent electron microscope studies will be presented separately. Correlation of the results of the immunofluorescence and electron microscope studies suggests the possibility that much of the immunofluorescence observed might be due to antigen in virus precursors or components.     

鸭胚成纤维细胞中鸭瘟强毒的增殖特性研究

通过细胞培养物光学显微观察、细胞超薄切片研究、定量PCR检测技术对鸭胚成纤维细胞中鸭瘟强毒的增殖特性进行了研究.结果表明:鸭瘟强毒接种鸭胚成纤维细胞后42 h,可使细胞培养物出现大量明显的蚀斑病变.细胞培养物的超薄切片电镜观察研究表明,病毒核酸在胞核内常集中分布,呈直径35～45 nm的圆形颗粒状;核衣壳在胞核和胞浆内都有分布,呈直径90～100 nm的网形颗粒状;成熟病毒位于胞浆空泡内,呈直径150～300 nm的圆形,有囊膜和皮层结构;病毒通过囊膜与胞膜融合入侵细胞,在核内生成核酸、装配核衣壳,在胞浆中得到皮层,出芽到胞浆空泡内获得囊膜,通过胞吐作用释放到胞外.定量PCR研究表明:鸭瘟强毒接种细胞后10 h开始明显复制,接毒后30 h时含量趋于稳定,接毒后22 h时开始向胞外释放,50 h时达最大值,细胞和上清中病毒含量的增幅均为103左右,病毒主要存在于细胞中,其含量为上清的102～103倍.     

Discovering new insect viruses: whitefly iridovirus (Homoptera: Aleyrodidae: Bemisia tabaci)

Adult whiteflies, Bemisia tabaci (Gennadius), collected from the field were screened for viral pathogens using a cell line from the silverleaf whitefly, B. tabaci, B biotype (syn. B. argentifolii). Homogenates from the field-collected whiteflies were applied to cell cultures and checked for cytopathic effects (CPE). Cells were observed to develop cytoplasmic inclusions and to have a change in morphology. Cells displaying CPE were observed using a transmission electron microscope and found to be infected with a virus. The virus particles had an icosahedral shape and an approximate size of 120-130 nm. The virus was observed in defined areas of the cytoplasm adjacent to the cell nucleus. Analysis using polymerase chain reaction, Southern blot hybridization, and DNA sequencing confirmed that the virus discovered infecting the whitefly cell cultures was an iridovirus. Sequence analysis showed that the amplimer (893 bp) had a 95% homology to the invertebrate iridescent virus type 6 major capsid protein gene. Discovery of new viruses of whiteflies may provide renewed interest in using pathogens in the development of innovative management strategies. This is the first report of an iridescent virus isolated from whiteflies, B. tabaci, collected from the field.