兔感染人巨细胞病毒AD169的初步探讨

目的 探讨兔对人巨细胞病毒（Human cytomegalovirus,HCMV）AD169毒株（静脉途径接种）的敏感性。方法 将HCMVAD169接种于体外培养的兔胚肺（Rabbit embryo lung,REL）细胞,观察病变,免疫组化检测病毒抗原,电镜观查细胞病毒颗粒。30只新西兰兔分别静脉途径接种1万个TCID50、100个TCID50、灭活HCMVAD169,观察动物发病情况、组织病理学变化和病毒DNA。结果 REL细胞在接种病毒后16h与HEL细胞同步开始出现细胞病变,免疫组化在接种病毒16h的细胞内查到HCMV特异抗原,在接种病毒36h的细胞核、浆内查到病毒颗粒。动物在接种病毒后7d出现病态表现,病情与接种量呈正相关。尸体解剖发现受累器官广泛,在多种组织细胞内可以查到HCMV基因。结论 HCMVAD169株在体外REL细胞中进行病毒复制。静脉途径接种HCVMAD169株可以感染家兔,通过控制病毒接种量可获得不同感染程度的模型。     

Experimental canine adenovirus glomerulonephritis: histological,immunofluorescence and ultrastructural features of the early glomerular changes.

Eighteen 16-week-old dogs were inoculated i.v. with a virulent strain of canine adenovirus and the renal glomeruli were examined by light, electron (transmission and scanning) and immunofluorescence microscopy at intervals from 24 h to 7 days after inoculation of virus. From 2 days onwards intranuclear inclusion bodies and cell-associated viral antigen were detected in mesangial, endothelial and circulating mononuclear cells. This was accompanied by swelling and, in some cases, necrosis of these cells with partial or complete occlusion of capillary loops. In 5 dogs examined on days 5-7 after inoculation of virus, granular deposition of IgG, C3 and viral antigen was observed in mesangial areas and electron dense deposits were found in the mesangial matrix.     

The replication of infectious bronchitis virus in fowl trachea

Summary Domestic fowl were exposed to an aerosol of infectious bronchitis virus. Birds were killed at intervals after infection and sections of trachea were examined in the electron microscope. The virus replicated in superficial cells,i.e., ciliated epithelial cells and mucus cells. Both these cell types were desquamated over large areas but the underlying basal cell layer was not significantly damaged. At an early stage of replication, virus particles were confined to a small vacuolated region of cytoplasm but in desquamated cells the whole cytoplasm was vacuolated and the vacuoles contained virus particles. Virus was not found in the nucleus and all cytoplasmic particles were within vacuoles. Virus formed by budding at vacuole membranes and the vacuoles involved were derived from both Golgi vesicles and endoplasmic reticulum. The virus probably has a linear internal component and is limited by a trilaminar membrane bearing projections which tend to stain lightly.Regeneration of morphologically normal tracheal epithelium occurred between 6 and 7 days after inoculation but a second wave of virus replication in the trachea was not detected although the virus could be recovered from inoculated birds for up to 14 days following inoculation.     

Neuromorphological and immunofluorescence study of experimental infection caused by the attenuated strain of tick-borne encephalitis virus]

In Syrian hamsters inoculated subcutaneously with an attenuated strain of tick-borne encephalitis virus the infection runs an asymptomatic course in all the animals, some of which, however, develop within 4 weeks some changes in nerve cells and hypertrophy of astrocytes. The viral antigen is detected by Coons' method in nerve cells and spleen cells. An intensive treatment of the hamsters with cyclophosphane 106 days after virus inoculation provoked no morphological lesions in the brain which could have been associated with virus activation. No virus antigen could be found in brain impression smears either. However, in both untreated animals and those treated with the immunosuppressant drug the viral antigen was found in mononuclear cells of the spleen.     

用协同凝集试验和SPA—固相免疫电镜技术检测EHFV抗原

本文建立了检测流行性出血热病毒(EHFV)抗原的协同凝集试验(COA)和SPA—固相免疫电镜技术(SPA—SPIEM)。COA检测粗制抗原时,先以正常鼠脑和肺悬液吸收用于致敏的抗血清,并用Cowanl株葡萄球菌预先处理抗原,可排除非特异性凝集反应。SPA—SPIEM似较COA敏感。研究结果提示COA和SPA—SPIEM均可用于EHFV抗原的检测,有可能被用于EHF疫区宿主流行病学调查和疾病的快速诊断。     

Pathogenesis of bronchiolitis and pneumonia induced in neonatal and weanling rats by parainfluenza (Sendai) virus.

The objective of this research was to identify potential mechanisms that might account for the greater susceptibility of neonatal rats (5 days old) as compared with weanling rats (25 days old) to viral-induced lung injury. Sites of viral replication, the sequential development of bronchiolitis and pneumonia, and systemic humoral immune responses were compared between neonatal and weanling rats from 2 to 17 days after being inoculated intranasally with parainfluenza Type 1 (Sendai) virus. In both neonatal and weanling rats, viral antigen was demonstrated by immunoperoxidase technique and viral nucleocapsids, and budding virions were demonstrated by transmission electron microscopy in bronchiolar ciliated and nonciliated epithelial cells as early as 2 days after inoculation. Similar evidence of viral replication was also demonstrated in both ages of rats in alveolar Type I and Type II epithelial cells and in macrophages. Neither virus nor viral antigens was identified in endothelial cells or interstitial cells of interalveolar septa. Bronchiolitis was induced as early as 2 days after inoculation in both ages of rats and was characterized by necrosis, erosion, and hyperplasia of epithelial cells. Multifocal to locally extensive interstitial pneumonia centered on bronchioles, was characterized by alveolar epithelial cell damage and by infiltration of neutrophils, macrophages, and lymphocytes in alveolar septa and spaces and was observed as early as 2 days after inoculation in both age groups of rats. Interstitial pneumonia of maximal severity occurred in weanling rats at 5 days after inoculation, whereas maximal pneumonia of comparable severity occurred in neonatal rats at 8 days after inoculation. Epithelial repair and resolution of bronchiolitis and pneumonia were largely complete in both ages of rats by 17 days after inoculation. Virus was recovered from lung homogenates of neonatal and weanling rats as early as 2 days after inoculation. In weanling rats, infective virus could not be recovered from lung beyond 6 days after inoculation, whereas in neonatal rats virus could be recovered from lung as late as 10 days after inoculation. Viral persistence in neonatal rats was associated with a delayed onset of serum antibody to the virus, compared with weanling rats. The results indicate that the cellular sites of viral replication and the pattern of inflammatory reactions are closely comparable between neonatal and weanling rats inoculated with Sendai virus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of HIV-1 in human thymic implant in SCID-hu mice after intravenous inoculation

Human immunodeficiency virus type 1 (HIV-1) was immunohistochemically and ultrastructurally localized in human thymus implants in SCID-hu mice 3 weeks after intravenous (i.v.) inoculation of the virus. A viral antigen (gp120) was predominantly distributed in and around the epithelial cells in Hassall's corpuscles as demonstrated by fluorescence immunohistochemistry. Occasional solitary round cells positive for the viral antigen but negative for cytokeratin were detected in the perivascular areas. Ultrastructural examinations clearly revealed a number of mature viral particles in the intercellular spaces of the Hassall's corpuscles. Thus the present study indicates the possibility that thymic epithelial cells in Hassall's corpuscles act as a target and/or reservoir in an early stage of HIV infection.     

Experimental Cytomegalovirus Ophthalmitis

Cytomegalovirus can produce a severe necrotizing chorioretinitis in patients on immunosuppressive therapy and infants born with congenital cytomegaloviral inclusion disease. To study the effect of cytomegalovirus on the eye, murine cytomegalovirus was injected into the eyes of nonimmunosuppressed Swiss CD-1 weanling mice. The eyes were then prepared for virus titer, as well as light and electron microscopy at variable periods after inoculation (1 to 28 days). From days 2 to 6, the hallmarks of cytomegalovirus infection, intranuclear and intracytoplasmic viral inclusions, were evident within cytomegalic cells. The major site of reaction was in the uveal tract, where necrosis and inflammation were prominent. Viral particles budding through the nuclear membranes into the perinuclear cisternae and vacuoles with viral particles could be seen in the cytoplasm of infected cells. In lesions older than 2 weeks, only a mild mixed inflammatory infiltrate and fibrosis were observed. Morphologic alterations unaccompanied by inflammation occurred in the outer sensory retina overlying infected retinal pigment epithelial cells. Multiple necrotic foci with inclusion-bearing cells in the liver indicated the systemic spread of virus from the eye. The titer of virus recovered from the eye peaked at day 4 and then declined to low levels, but infectious virus could still be isolated at day 28, even though viral particles were not seen morphologically at or after day 14. Many of the alterations seen in the model resemble those found in the human cytomegaloviral ophthalmitis.     

Attenuated variants of eastern equine encephalomyelitis virus: pathomorphological, immunofluorescence and virological studies of infection in Syrian hamsters.

A virulent strain of eastern equine encephalomyelitis virus produced in hamsters a lethal infection with severe lesions of nerve cells predominatly in the anterior parts of the brain. Parenchyma necroses occurred in the liver and lymphoid organs. Infectious virus and viral antigen were found in all the organs examined with the greatest accumulation in the brain. Attenuated variants of the virus produced in most hamsters an inapparent infection. In some animals without clinical signs, focal inflammatory-dystrophic lesions were found in the central nervous system (CNS) and the liver, as were immunomorphological changes in the lymphoid tissue. In the latter infectious virus could be detected for 6 days after inoculation (p.i.), whereas in the brain only viral antigen could be found up to 14 days. At 3 and 6 months p.i., no persistence of attenuated virus in the brain and lymphoid tissues could be established by organ culture and co-cultivation methods. Nor was virus antigen detected. No pathomorphological changes or proliferative-hypertrophic astrocyte reaction were found.     

Biogenesis of Marek''s Disease (Type II Leukosis) Virus In Vitro: Electron Microscopy and Immunological Study

The kinetic events involved in Marek's disease herpesvirus infection of avian cell culture were investigated by assaying viral infectivity and antigenicity as well as by electron microscopy during the infectious cycle. The levels of viral infectivity and complement-fixing (CF) antigens revealed that the rates of appearance of infectious particles and CF antigens were not synchronous. Viral specific CF antigen could be detected 5 h after infection, whereas viral infectivity or the appearance of viral particles could be demonstrated only after 10 h of infection. High proportions of the recovered CF antigens during the various stages of the infectious cycle were found to be soluble and did not sediment with the virus particles. Cytological analysis of the developmental stages of the JM virus-infected cells by thin sectioning and electron microscopy revealed that at 8 h small particles approximately 35 nm in diameter appeared in the cell nuclei. The appearance of nucleocapsids occurred at 10 h, and these were of varying shapes; however, all were approximately 100 nm in diameter. At approximately 18 h postinfection, mature virus particles were observed. Viral maturation of the immature particles occurred by the acquisition of envelope from the inner leaflet of the nuclear membrane or from the cytoplasmic membrane of the cell.     

Development of the infection in oat leaves inoculated with barley yellow dwarf virus.

Electron microscopic examination of oat (Avena sativa L.) leaves inoculated with barley yellow dwarf virus revealed the presence of the following inclusions in phloem parenchyma, companion cells and sieve elements: Viruslike particles, slender filaments, small vesicles containing fibrils, and an amorphous material. Cells with inclusions, presumed to be infected cells, were detected as early as 2 days after inoculation. At 4 days, virus particles and filaments were observed in some mature sieve elements. A densely staining material and also, occasionally virus particles were seen in many plasmodesmata connecting infected mature elements with adjacent immature sieve elements. Within the latter, accumulations of filaments, or filaments and virus particles, with amorphous material were found in the cytoplasm centered around the plasmodesmatal entrances and occasionally in the interior of the cells. Small vesicles containing fibrils usually occurred around the periphery of the accumulations and, more frequently, at and near the tonoplast. Early in the infection of the cell, the nucleus became distorted and filaments resembling those associated with the virus particles in the cytoplasm appeared in the nucleoplasm and nuclear pores. At a later stage the heterochromatin became clumped and virus particles were observed in the nucleoplasm. At 6 and 11 days, inclusions were seen in almost all phloem cells. Passage of the virus particles and filaments from many of these cells into channels of translocation was suggested by the appearance of these inclusions in plasmodesmata and in adjacent sieve elements. The possible nature of the unusual filamentous inclusions is discussed.     

Okra mosaic virus empty protein shells in nuclei

Okra mosaic virus produces large quantities (approximately 10 mg/g of tissue) of empty viral protein shells in inoculated cucumber cotyledons. Virus particles could not be detected in isolated nuclei, but empty protein shells accumulated there. At one day after inoculation, about half the total viral protein shells were found in the nucleus. This accumulation occurred in the presence of virus particles in the cytoplasm. The most likely explanation for this active and preferential accumulation is that viral coat protein enters the nucleus in the form of monomers or pentamers and hexamers, and is assembled into shells once inside.     

Leukocyte subpopulations in kidney and trachea of chickens infected with infectious bronchitis virus

Using immunohistochemical methods, we studied the nephropathogenicity of the infectious bronchitis virus (IBV)-strain V1648- and the leukocyte phenorypes in the pathological lesions in the kidneys and the trachea formed after inoculation with this virus strain. One-day-old WLA chickens were intravenously inoculated, and after 5, 7 and 11 days their kidneys, trachea and lungs were removed. Monoclonal antibodies were used to detect viral antigen, and lymphoid and non-lymphoid cell populations. In serial sections, the detection of the viral antigen was correlated to the phenotypes of the cells. At days 5 and 7 after inoculation, viral antigen was detected in the epithelium and the interstitium of the kidney tubuli and in the epithelium of the trachea. The infiltrated cells in these tissues were mainly of the T cell phenotype. The cellular immune reaction was correlated with the detection of viral antigen.     

C-type virus-lymphocyte interactions in developing mouse thymus

The appearance of C-type virus particles in thymus cells of Swiss mouse embryos, 11.5 to 15.5 days post-conception age (PCA), was studied with the electron microscope. In thymic rudiments of all specimens examined, virus particles were seen in epithelial cytoplasm, budding from epithelial cell surfaces and in extracellular spaces. Lymphoid cells were first seen in thymic rudiments of 13.5 days PCA, and did not display virus particles at this stage. At 14.5 days PCA, thymic lymphocytes had localized plasmalemmal thickenings of high electron-density which were adjacent to extracellular virus particles. Viruses appeared to be penetrating thymic lymphocytes by viropexis in embryos of 15.5 days PCA. At this stage, many lymphocytes also had cytoplasmic virus-containing vesicles and viral buds at their surfaces. These observations suggest the possibility that, in embryos, C-type I viruses are transmitted horizontally from thymic epithelium to early populations of thymic lymphocytes.     

Sequential development of antigens of equine rhinopneumonitis virus in cultured horse kidney cells as studied with fluorescent antibodies

Summary The development of V and S antigens in horse kidney cells infected with equine rhinopneumonitis virus was studied.During the latent period, S antigen was first detected and developed exclusively in the nucleus, whereas V antigen appeared in the cytoplasm with increase in intracellular infectivity. As the virus multiplication proceeded, the whole cell was filled with both antigens, followed by the disappearance of antigenic materials from the cells except the peripheral area at the terminal stage of infection. The development of inclusion materials as revealed by hematoxylin and eosin staining was closely related to the increase of virus titer as well as the development of the viral antigens except at the late stages of infection.These observations together with those by electron microscopy and other methods suggest that in the infected cells S antigen is synthesized in the nucleus, followed by the synthesis of V antigen in the cytoplasm and finally the mature virus particle is completed in the cytoplasm by assembly of both antigenic materials, and that S-antigen represents DNA-protein constituting the viral core and V antigen constitutes the virus particle surface of protein nature.     

Ultrastructural localization by immunoperoxidase techniques of influenza virus antigens in abortive infection of L cells

An abortive infection was induced in L cells by influenza virus A/Hong Kong/68 (H3N2). With the use of antibody and peroxidase-labelled protein A, the localization of virus protein synthesis but not the maturation of virus particles was demonstrated at the ultrastructural level. Five days after inoculation (p.i.), the synthesis of viral haemagglutinin was localized in the region of the rough endoplasmic reticulum; at late intervals p.i., haemagglutinin accumulated in the plasma membranes, where membrane vesicles, containing haemagglutinin in their membranes, were released from the cell surface. The cytoplasmic viral ribonucleoprotein was localized in the region of free cytoplasmic ribosomes and that of the outer sheet of the nuclear membrane. Viral proteins were detected in the cytoplasm and plasma membranes also after 70 and 390 days of passaging of the cells or of their long-term cultivation with regular change of medium.     

Pathogenesis of haemagglutinating encephalomyelitis virus (HEV) in mice experimentally infected by different routes

Three-day-old suckling mice inoculated intracerebrally (i.c.) with the 67N strain of haemagglutinating encephalomyelitis virus (HEV) showed nervous signs and died. The virus was passaged 10 times in suckling mice and was designated the MB-67N strain. The pathogenesis of MB-67N was studied with various ages of mice and inoculation routes. All mice inoculated i.c. with a large dose of virus died regardless of age, although a smaller dose caused fatal infection only in suckling mice. By intranasal, intraperitoneal and subcutaneous inoculation, the virus also killed suckling mice under 16 days old, but not older mice, even with a large dose. The susceptibility of mice for the MB-67N strain was influenced by age and inoculation routes. High titres of virus were re-isolated from the brain of diseased mice after inoculation by any route, but not from other organs. Histologically, numerous areas of severe focal necrosis were produced in the cerebral cortex. Specific immuno-fluorescence and numerous viral particles were found in the cytoplasm of nerve cells by immuno-fluorescence staining and electron microscopy. These findings indicate that the MB-67N propagates mainly in the central nervous system and nerve cells serve as a main target of virus replication.     

Tubular structures in the cervix of mice experimentally infected with herpes simplex virus type 2

Experimental infection of the C3H/N mouse genital tract was demonstrated after intravaginal inoculation with herpes simplex virus type 2 (HSV-2). About 75% of the infected animals died by Day 7, and 75% of the surviving animals had severe vaginitis or neurological signs on Day 7. Titers of the virus recovered from vaginal secretions of infected animals reached a maximum on Day 2 and gradually decreased until Day 7. On the other hand, under the electron microscope, virus particles and tubular structures could be found in the nuclei of infected cells of the cervix in the 1st, 2nd and 4th days after infection. All cases in which virus particles could be found in the nuclei of infected cells were also positive for tubular structures and vice versa. These observations indicate that in situ diagnosis of HSV-2 infection can be made in the mouse model. The same method would be applicable for the diagnosis of human HSV-2 infection.     

Morphology of adenovirus type-3 infection of human respiratory epithelial cells in vitro

The adenovirus is a non-enveloped DNA virus which may lead to severe diseases of the respiratory tract. In order to study the influence of virus infection on primary cultured peribronchial submucosal gland cells, we performed in vitro infection with human adenovirus type 3. Peribronchial submucosal glands are the main source of tracheobronchial mucus and, therefore, play a major pathophysiological role in common pulmonary diseases such as bronchial asthma, chronic obstructive pulmonary disease and cystic fibrosis. The success of infection was verified by means of immunofluorescence and transmission electron microscopy. Infection follows a certain timetable with a climax of paracristalline intranuclear virus inclusions after 48 h of infection. Virus particles could be detected in the nucleus as well as in peripheral and perinuclear cytoplasmatic vacuoles. The release of virus capsids from the nucleus could be visualized using transmission electron microscopy and immunofluorescence with antibodies against hexon proteins. Two different kinds of mechanisms of transition of newly synthesized virus capsids from the nucleus into the cytoplasm could be identified. Due to an increasing cytopathic effect, viruses spread from cytoplasm after longer terms of infection. Cytopathic effects and cytoskeleton aspects under this virus infection could be characterized using immunofluorescence with several monoclonal antibodies against different cytokeratins.