鸭病毒性肠炎病毒强毒株的形态发生学与超微病理学研究

应用透射电镜和超薄切片技术,研究鸭病毒性肠炎病毒(duck enteritis virus,DEV)CH强毒株人工感染成年鸭后,病毒在宿主细胞内的形态发生及各组织器官的超微结构变化.结果表明,感染后不同时间剖杀及发病后死亡鸭的肝、肠、脾、胸腺、法氏囊等组织器官中,均观察到典型的疱疹病毒粒子.病毒主要的靶细胞为淋巴细胞、网状内皮细胞、成纤维细胞、巨噬细胞、血管内皮细胞、肠道上皮细胞、肠道平滑肌细胞和肝细胞等.DEV的核衣壳有空心型、致密核心型、双环型和内壁附有颗粒型四种形态,存在胞核和胞浆两种装配方式.病毒核衣壳可在核内获得皮层,通过核内膜获得囊膜成为成熟病毒;也可通过内外核膜进入胞浆,在其中获得皮层,然后在各种质膜上获得囊膜,最后成熟病毒释放到细胞外.伴随着病毒的复制、装配和成熟,细胞中出现多种核内和胞浆包涵体、核内致密病毒核酸颗粒、微管和中空短管以及胞浆内膜包裹的电子致密小体、双层管等病毒相关结构.超微研究表明,组织细胞有坏死和凋亡两种变化.坏死细胞肿胀甚至破裂,线粒体肿胀空泡化,粗面内质网扩张,核糖体脱落,有的细胞器甚至完全崩解,染色质或固缩或溶解.凋亡细胞则染色质聚集,胞浆凝聚深染,细胞膜上有大量空泡,并有凋亡小体形成.细胞坏死与凋亡往往同时存在,疾病发生过程中,脾、胸腺、法氏囊以及小肠固有层中的淋巴细胞凋亡数量明显增多.     

Cell-Free Transmission and In Vivo Replication of Marek''s Disease Virus

Marek's disease virus recovered from the feather follicle of infected chickens was found to be infectious for chickens in cell-free preparations. The virus replicated in epithelial cells of the germinative layer of the feather follicle epidermis, producing both intranuclear and round or diffuse cytoplasmic inclusion bodies in the infected cells. It was found at this site 2 weeks postinoculation and prior to the development of tumor or other gross lesions. In the nucleus, many naked and a few enveloped herpesvirions were found, whereas the cytoplasm contained predominantly enveloped herpesvirions, which were usually within the cytoplasmic inclusion bodies. Approximately 80% of the extracellular virions were enveloped. Studies with both virulent and avirulent strains of the virus revealed a relationship between virulence, contagiousness, and replication of the virus in the feather follicle.     

Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.     

The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection.

To demonstrate the essential nature of the baculovirus GP64 envelope fusion protein (GP64 EFP) and to further examine the role of this protein in infection, we inactivated the gp64 efp gene of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and examined the biological properties of this virus in vivo. To provide GP64 EFP during construction of the recombinant GP64 EFP-null AcMNPV baculovirus, we first generated a stably transfected insect cell line (SfpOP64-6) that constitutively expressed the GP64 EFP of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The AcMNPV gp64 efp gene was inactivated by inserting the bacterial lacZ gene in frame after codon 131 of the gp64 efp gene. The inactivated gp64 gene was cloned into the AcMNPV viral genome by replacement of the wild-type gp64 efp locus. When propagated in the stably transfected insect cells (Sf9OP64-6 cells), budded virions produced by the recombinant AcMNPV GP64 EFP-null virus (vAc64z) contained OpMNPV GP64 EFP supplied by the Sf9OP64-6 cells. Virions propagated in Sf9OP64-6 cells were capable of infecting wild-type Sf9 cells, and cells infected by vAc64z exhibited a blue phenotype in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). Using cytochemical staining to detect vAc64z infected cells, we demonstrated that this GP64 EFP-null virus is defective in cell-to-cell propagation in cell culture. Although defective in cell-to-cell propagation, vAc64z produces occlusion bodies and infectious occlusion-derived virions within the nucleus. Occlusion bodies collected from cells infected by vAc64z were infectious to midgut epithelial cells of Trichoplusia ni larvae. However, in contrast to infection by a control virus, infection by vAc64z did not proceed into the hemocoel. Analysis of vAc64z occlusion bodies in a standard neonate droplet feeding assay showed no virus-induced mortality, indicating that occluded virions produced from vAc64z could not initiate a productive (lethal) infection in neonate larvae. Thus, GP64 EFP is an essential virion structural protein that is required for propagation of the budded virus from cell to cell and for systemic infection of the host insect.     

Morphogenesis of Sindbis virus in cultured Aedes albopictus cells.

Cultured mosquito cells were found to produce Sindbis virus nearly as efficiently as BHK-21 cells at 28 C. In virtually all of the cells observed in the electron microscope, virus morphogenesis was found to occur within complex vesicular structures which developed after viral infection. Viral nucleocapsids were first seen in these vesicles and appeared to be enveloped within these structures. The process of envelopment within these inclusions differed in some respects from the process previously described for the envelopment of nucleocapsids at the plasma membrane of vertebrae cells. Free nucleocapsids were only rarely seen in the cytoplasm of infected mosquito cells, and budding of virus from the cell surface was detected so infrequently that this process of virus production could not account for the amount of virus produced by the infected cells. The vast majority of extracellular virus was produced by the fusion of the virus-containing vesicles with the plasma membrane releasing mature virions and membrane nucleocapsid complexes in various stages of development.     

Herpes simplex virus type 1 primary envelopment: UL34 protein modification and the US3-UL34 catalytic relationship

The herpes simplex virus type 1 (HSV-1) US3 kinase is likely important for primary envelopment of progeny nucleocapsids since it localizes to the nuclear envelope of infected cells and largely determines the phosphorylation state and localization of the necessary primary envelopment factor, the UL34 protein. In HEp-2 cells, the production of infectious US3 null progeny is delayed and decreased relative to that of the parental strain, HSV-1(F). Furthermore, the US3 kinase affects the morphology of primary envelopment such that in its absence, UL34 protein-containing enveloped virions accumulate within membrane-bound vesicles. These vesicles are most often found along the interior periphery of the nucleus and may be derived from the inner nuclear membrane. Since the US3 and UL34 proteins comprise a kinase-substrate pair, a reasonable hypothesis is that the US3 kinase influences these replication parameters by direct phosphorylation of the UL34 protein. For this report, recombinant viruses were constructed to determine the significance of UL34 protein phosphorylation and US3 catalytic activity on UL34 protein localization, single-step growth, and envelopment morphology in both HEp-2 and Vero cells. The data presented suggest that the significance of UL34 phosphorylation is cell type dependent and that efficient viral morphogenesis requires US3-mediated phosphorylation of an infected cell protein other than UL34.     

大菜粉蝶颗粒体病毒的形态发生及细胞病理的超微结构观察

本文叙述感染大菜粉蝶颗粒体病毒后,病虫脂肪体细胞超微结构的改变,大菜粉蝶感染后24小时,病虫脂肪体细胞开始出现明显的病变,整个病程是,在开始时细胞核内出现清晰区并出现病毒发生基质,核膜多点成套增生,其后核膜断裂,大量膜样结构聚集在病毒发生基质的周围,核衣壳大量产生,有一部分核衣壳从这些病毒发生基质四周的膜样结构碎片上获得套膜,荚膜蛋白沉积形成成熟的病毒荚膜,或称包含体;另一部分则排列在胞浆内的空泡边缘上;其余的核衣壳则从细胞边缘“芽突”而获得套膜,另外还描述环孔片层及线粒体改变。     

AcMNPV Core Gene ac109 Is Required for Budded Virion Transport to the Nucleus and for Occlusion of Viral Progeny

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac109 core gene has been previously characterized as an essential late gene. Our results showed that budded virions could be detected in supernatants of infected Sf-9 cells, even when ac109 knockout viruses displayed a single-cell infection phenotype. Moreover, confocal microscopy analysis revealed that budded virions can enter the cytoplasm but are unable to enter the cell nucleus. This defect could be repaired by complementing ac109 in trans. In addition, polyhedra of normal size could be detected in Sf-9 nuclei infected with ac109 knockout viruses. However, electron microscopy demonstrated that these occlusion bodies were empty. Altogether, these results indicate that ac109 is required for infectivity of both phenotypes of virus.     

Autographa Californica Multiple Nucleopolyhedrovirus P48 (Ac103) Is Required for the Efficient Formation of Virus-Induced Intranuclear Microvesicles

Our previous study has shown that the Autographa californica multiple nucleopolyhedrovirus(AcMNPV) p48(ac103)gene is essential for the nuclear egress of nucleocapsids and the formation of occlusion-derived virions(ODVs). However,the exact role of p48 in the morphogenesis of ODVs remains unknown. In this study, we demonstrated that p48 was required for the efficient formation of intranuclear microvesicles. To further understand its functional role in intranuclear microvesicle formation, we characterized the distribution of the P48 protein, which was found to be associated with the nucleocapsid and envelope fractions of both budded virions and ODVs. In Ac MNPV-infected cells, P48 was predominantly localized to nucleocapsids in the virogenic stroma and the nucleocapsids enveloped in ODVs, with a limited but discernible distribution in the plasma membrane, nuclear envelope, intranuclear microvesicles, and ODV envelope. Furthermore,coimmunoprecipitation assays showed that among the viral proteins required for intranuclear microvesicle formation, P48 associated with Ac93 in the absence of viral infection.     

Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR

The Koi herpesvirus (KHV) is a herpes-like virus now recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp Cyprinus carpio carpio. Temperature is a key factor influencing virus replication both in cell culture and in the tissues of experimentally infected fish. Genomic DNA sequences were used to optimize a rapid real-time TaqMan PCR assay to detect and quantify KHV DNA as found in the tissues of virus-exposed fish. The assay allowed analytical enumeration of target KHV genome copies ranging from 10(1) to 10(7) molecules as present in infected cell lines or fish tissues. The new assay was specific for KHV and did not detect DNA from 3 related herpes-like viruses found in fish, the Cyprinid herpesvirus 1 (CyHV-1), Cyprinid herpesvirus 2 (CyHV-2), Ictalurid herpesvirus 1 (IcHV-1) or the KF-1 cell line used for virus growth. Concentrations of KHV DNA were evaluated in 7 different tissues of replicate groups of virus-exposed koi held at water temperatures of 13, 18, 23 and 28 degrees C. Viral DNA was detected among virus-exposed koi at all 4 water temperatures but mortality was only observed among fish at 18, 23, and 28 degrees C. Time and temperature and the interactions between them affected concentrations of viral DNA detected in tissues of koi exposed to KHV. Although there were no recognized patterns to viral DNA concentrations as found in different tissues over time, KHV genome copies for all tissues increased with time post virus exposure and with water temperature. The remarkably rapid and systemic spread of the virus was demonstrated by the presence of viral DNA in multiple tissues 1 d post virus exposure. The greatest DNA concentrations found were in the gill, kidney and spleen, with virus genome equivalents consistently from 10(8) to 10(9) per 10(6) host cells. High levels of KHV DNA were also found in the mucus, liver, gut, and brain. Koi surviving infection at 62 to 64 d post virus exposure contained lower KHV genome copies (up to 1.99 x 10(2) per 10(6) host cells) as present in gill, kidney or brain tissues.     

水痘-带状疱疹病毒地方分离株在兔脑神经细胞中的形态与形态发生特征

为阐明水痘-带状疱疹病毒济南分离株（VZVJ1）在兔脑神经细胞（RNC）中的形态与形态发生特征,我们利用超薄切片电子显微镜技术对感染VZVJ1的RNC进行了观察研究。结果表明RNC在感染VZVJI6h后核内可见散在的核衣壳,12h后细胞核和细胞质内核衣壳明显增多,24h达高峰,而细胞核和细胞质内的成熟病毒颗粒较少见。病毒大小、形态基本一致,呈圆形或椭圆形,核心直径30～50nm,核衣壳74～96nm,成熟病毒110～180nm。核衣壳内有3种类型的核心,即电子致密核心、部分致密核心和电子透明核心。细胞核和细胞质内均可见核心样电子致密体和布纹样结构。在细胞质内还可见少量“繁殖复合体”,由膜性结构包绕多个囊泡构成。提示VZVJ1在RNC中的形态发生不同于其它性质的细胞。     

Histopathology of a nuclear polyhedrosis infection in Aedes epactius with observations in four additional mosquito species

Aedes epactius larvae were utilized to study the infection sequence of the nuclear polyhedrosis virus (NPV) from Aedes sollicitans. From 30 min to 6 hr postinoculation, polyhedra and many free virions were observed in the larval midgut lumen. Penetration of the midgut cells by virions was not observed. The first infected nuclei were observed 12 hr postinoculation. Nucleocapsids initially exhibited electron translucent cores which became electron dense before the nucleocapsids acquired an envelope. Envelope acquisition occurred through a process of de novo membrane morphogenesis. Occlusion of the singly embedded virions began by 18 hr postinoculation with the mature rough-surfaced polyhedra averaging approximately 1 by 2 μm. Unusually long nucleocapsids (approximately two or three times the length of other nucleocapsids) were only observed in late infection period nuclei. There was no evidence that long nucleocapsids represented an early developmental stage for nucleocapsids of standard length. Infection was restricted to midgut nuclei and gastric caecae cells. Infected early instar A. epactius larvae became moribund 36 to 40 hr postinoculation and infected midgut nuclei were observed to undergo lysis. The late stages of NPV infection were observed in larvae of A. annandalei, Wyeomyia smithii, Toxorhynchites brevipalpus, and Eretmapodites quinquevittatus. Virion development and occlusion in these species was basically identical to the sequence observed in A. epactius larvae.     

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.     

Characterization of two Autographa californica nucleopolyhedrovirus proteins, Ac145 and Ac150, which affect oral infectivity in a host-dependent manner

The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) contains two homologues, orf145 and orf150, of the Heliothis armigera Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this protein within the nucleus in areas of nucleocapsid assembly and maturation, along with some association with the enveloped bundles of virions within the developing occlusion bodies (OBs). Ac150 was found to be mainly associated with enveloped bundles of virions within OBs and also with those not yet occluded. Both Ac145 and Ac150 were found to be present in budded virus as well as OBs. Both orf145 and orf150 were deleted from the AcMNPV genome, singly or together, and these deletion mutants were assessed for oral infectivity both in Trichoplusia ni and Heliothis virescens larvae. Deletion of Ac145 led to a small but significant drop in infectivity (sixfold) compared to wild-type (wt) AcMNPV for T. ni but not for H. virescens. Deletion of Ac150 alone had no effect on infectivity of the virus for either host. However, deletion of both Ac145 and Ac150 gave a recombinant virus with a drastic (39-fold) reduction in infectivity compared to wt virus for H. virescens. Intrahemocoelic injection of budded virus from the double-deletion virus into H. virescens larvae is as infectious to this host as wt budded virus, indicating that Ac145 and Ac150 play a role in primary oral infection of AcMNPV, the extent of which is host dependent.     

Autographa californica multiple nucleopolyhedrovirus EXON0 (ORF141) is required for efficient egress of nucleocapsids from the nucleus

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) exon0 (orf141) has been shown to be required for the efficient production of budded virus (BV). The deletion of exon0 reduces the level of BV production by up to 99% (X. Dai, T. M. Stewart, J. A. Pathakamuri, Q. Li, and D. A. Theilmann, J. Virol. 78:9633-9644, 2004); however, the function or mechanism by which EXON0 affects BV production is unknown. In this study, we further elucidated the function of EXON0 by investigating the localization of EXON0 in infected Sf9 cells and in virions and by identifying interactions between EXON0 and other viral proteins. In addition, electron microscopy was used to study the cellular localization of nucleocapsids in cells transfected with an exon0 knockout (KO) virus. The results showed that EXON0 was localized to both the cytoplasm and the nuclei of infected Sf9 cells throughout the infection. Western blotting results also showed that EXON0 was purified along with BV and occlusion-derived virus (ODV). The fractionation of BV into the nucleocapsid and envelope components showed that EXON0 localized to the BV nucleocapsid. Yeast two-hybrid screening, coimmunoprecipitation, and confocal microscopy revealed that it interacted with nucleocapsid proteins FP25 and BV/ODV-C42. Cells transfected with the exon0 KO virus exhibited normally appearing nucleocapsids in the nuclei in numbers equal to those in the nuclei of cells transfected with the EXON0 repaired virus. In contrast, the numbers of nucleocapsids in the cytoplasm of cells transfected with the exon0 KO virus were significantly lower than those in the cytoplasm of cells transfected with the repaired virus. These results support the conclusion that EXON0 is required in the BV pathway for the efficient egress of nucleocapsids from the nucleus to the cytoplasm.     

Nonlytic spread of naked viruses

How do viruses spread from cell to cell? Enveloped viruses acquire their surrounding membranes by budding: either through the plasma membrane or an internal membrane of infected cells. Thus, a newly budded enveloped virus finds itself either in the extracellular milieu or in a lumenal compartment from which it can exit the cell by conventional secretion. On the other hand, naked viruses such as poliovirus, nodavirus, adenovirus, and SV40 lack an external membrane. They are simply protein-nucleic acid complexes within the cytoplasm or nucleus of the infected cell, and thus would seem to have no other exit route than cell lysis. We have presented the first documentation of nonlytic spread of a naked virus, and showed the interconnections between this event and the process or components of the autophagy pathway.