急性致死性对虾病的杆状病毒病原研究

近年来,上海郊区饲养中国对虾（Ｐｅｎａｃｕｓ ｃｈｉｎｅｓｉｓ ｏｓｂｅｃｋ）的虾塘发生大面积的流行病。经对病虾的鳃、肝、胰腺或中肠组织进行超薄切片处理及电镜观察。发现在这些组织的细胞核内存在大量有囊膜包裹的杆状病毒,有时一个囊膜还可同时包裹两个病毒的核衣壳。经有机溶剂处理、差速离心及密度梯度离心,可以获得部分提纯的病毒和病毒核衣壳制剂。超薄切片中,病毒粒子大小约为４４０－５００×１７０－１８０ｎ     

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.     

Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with white-spot syndrome virus

This study focused on apoptosis in various tissues of the black tiger shrimp Penaeus monodon following white spot syndrome virus (WSSV) injection. The study included: (1) light microscopy (LM) and transmission electron microscopy (TEM) of various tissues; (2) fluorescent LM of nuclear DNA by staining with 4, 6-diamidine-2-phenyl indole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labelling (TUNEL) techniques; and (3) determination of caspase-3 activity. Juvenile P. monodon were injected with WSSV, and several tissues of ectodermal and mesodermal origin were studied at different intervals after injection. The total haemocyte count had decreased to one-tenth of its original level 60 h after WSSV injection. By LM, extensive destruction by WSSV was observed in the stomach epithelium, gills, hematopoietic tissue, hemocytes and the heart, but the most severely affected tissue was the subcuticular epithelium. TEM revealed that at 6 h post-injection (p.i.) the chromatin of infected nuclei was marginated, and by 24 h p.i. the nuclei were filled with enveloped and non-enveloped WSSV virions. At later stages of the infection, the nucleus extruded WSSV particles. Chromatin margination and nuclear condensation and fragmentation (i.e. signs of apoptosis) were observed as early as 6 h p.i. in all affected tissues, but occurred in cells without WSSV virions rather than in cells with virions. The occurrence of apoptosis was supported by data obtained using TUNEL and by DAPI-staining and progressed from 6 to 60 h p.i. In addition, caspase-3 activity in WSSV-infected shrimp was about 6-fold higher than that in uninfected shrimp. The data strongly suggests that apoptosis occurs following WSSV infection in P. monodon, but the extent to which it contributes to shrimp mortality requires further investigation.     

Pathology and ultrastructure of an intranuclear bacilliform virus (IBV) infecting brown shrimp Crangon crangon (Decapoda: Crangonidae)

The brown shrimp Crangon crangon supports an important fishery in Europe (over 25000 t, valued at 80 million euros in 2000). Through the course of histopathological screening of crustaceans from the Clyde estuary, western Scotland, for the biological effect of contaminants, we have discovered a highly prevalent (up to 100%) non-occluded intranuclear bacilliform virus (IBV) infection in the hepatopancreatic tubule epithelia and midgut epithelia of wild C. crangon. This is the first report of an IBV in this family. We have termed this virus Crangon crangon bacilliform virus (CcBV). Histological and ultrastructural observations suggest that this virus is similar to other IBVs previously described from crabs and penaeid shrimps. The nuclei of virus-infected epithelial cells contained an eosinophilic, hypertrophied viroplasm that marginalised the chromatin of the host nucleus. Infected cells were often separated from their neighbouring cells and their nuclei appeared apoptotic. In heavily infected shrimp, apoptotic cells were expelled into the lumen of the hepatopancreatic tubule or the midgut. Following this stage, some hepatopancreatic tubules became degenerate, with remnants of the basement membrane and myoepithelial lining remaining. Transmission electron microscopy of hypertrophic nuclei revealed the presence of rod-shaped and cylindrical, envelope-bound virions. These virions did not form arrays and were not embedded within occlusion bodies, but did appear to be partially occluded in an amorphous matrix that corresponded to a granular viroplasm. The ultrastructure, morphology and size of the nucleocapsid and the complete virion aligns the virus most closely to the IBVs previously reported from other decapod crustaceans. Due to the pathological manifestation of IBV infection in C. crangon, it appears likely that it can act as a population modulator, particularly at sites where infection prevalence is high, such as that observed in the Clyde estuary.     

Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection

An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue-green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e-124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV.     

Ultrastructural identification of a herpes-like virus infection in common carp Cyprinus carpio in Korea

We report on a herpes-like virus, which was found to be associated with mass mortality of common carp Cyprinus carpio for the first time in Korea in 1998. The external signs of infection in moribund fish were darkened coloration and severe branchial necrosis in the gill. Transmission electron microscopy revealed the presence of herpes-like viruses in spleen tissue. Infected spleen cells showed hypertrophied nuclei and degeneration. Numerous nucleocapsids of about 82 nm in diameter were found within the nucleus and cytoplasm of infected cells, and extracellular enveloped particles were also observed. We conclude that this virus was a likely significant cause of the high mortality of common carp in Korea in 1998.     

Diseases of the eye of farmed shrimp Penaeus monodon

Lesions were found in the eyes of cultured shrimp Penaeus monodon that displayed non-specific signs of disease, including lethargy, dark pigmentation, brown gills, empty midgut, anorexia, white tail muscle, necrosis of uropods and fouled cuticle. Eye lesions were associated with sexual development in moribund shrimp in at least 1 disease event. Suppurative inflammation, granuloma and malacia were observed in histological examination of the eye and the causative agents of lesions appear to be Vibrio spp. and a rod-shaped virus (similar to Lymphoid Organ Virus, Gill-Associated Virus [GAV] and Yellow-Head Virus). Suppurative inflammation was characterised by edema, infiltration of haemocytes and local sites of abscesses. Eyes with granuloma usually appeared white in pond-side examinations, and histology showed that fibrous tissue replaced ommatidia, ganglia and internal structures of the eye. Malacia of the eye was characterised by necrosis of nervous tissue, vacuolation and vascular proliferation in the medulla ganglia. Levels of presumptive Vibrionaceace were high in moribund specimens and Gram-negative rods were observed in some specimens as free particles in the interstitial fluid and haemolymph in the eye. Transmission electron microscopy showed that nerve cells in the fasciculated zone (near the basement membrane) contained cytoplasmic vesicles (1 to 3 microm in diameter) with particles (15 to 26 nm in diameter) and rod-shaped nucleocapsids. The rods were similar to those of GAV and were 130 to 260 nm long, 10 to 16 nm in diameter and had helical symmetry with a screw-like thread (2.4 to 3.5 nm pitch). Also, unidentified enveloped virions, averaging 74 nm in diameter, were observed in cytoplasmic vesicles in the fasciculated zone. In conclusion, it is suggested that bacterial and viral infections of the eye could result in impaired neuroendocrine functions, which may cause a range of clinical signs of disease.     

White spot syndrome virus isolates of tiger shrimp Penaeus monodon (Fabricious) in India are similar to exotic isolates as revealed by polymerase chain reaction and electron microscopy

Microbiological analysis of samples collected from cases of white spot disease outbreaks in cultured shrimp in different farms located in three regions along East Coast of India viz. Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa), revealed presence of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas spp. but experimental infection trials in Penaeus monodon with these isolates did not induce any acute mortality or formation of white spots on carapace. Infection trials using filtered tissue extracts by oral and injection method induced mortality in healthy P. monodon with all samples and 100% mortality was noted by the end of 7 day post-inoculation. Histopathological analysis demonstrated degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ with presence of intranuclear basophilic or eosino-basophilic bodies in tubular cells and intercellular spaces. Analysis of samples using 3 different primer sets as used by other for detection of white spot syndrome virus (WSSV) generated 643, 1447 and 520bp amplified DNA products in all samples except in one instance. Variable size virions with mean size in the range of 110 x 320 +/- 20 nm were observed under electron microscope. It could be concluded that the viral isolates in India involved with white spot syndrome in cultured shrimp are similar to RV-PJ and SEMBV in Japan, WSBV in Taiwan and WSSV in Malaysia, Indonesia, Thailand, China and Japan.     

利用对虾原代细胞增殖对虾杆状病毒HHNBV的研究

自1993年起,国内养殖的中国对虾(Ｐｅｎａｅｕｓｃｈｉｎｅｎｓｉｓ)出现暴发性流行病。该病来势猛,发病急,传播快,死亡率高,严重影响我国的对虾养殖。在青岛地区,发病时间在6～8月。一旦发病,全池对虾可在3～10ｄ内大部或全部死亡。现已查明,该流行病的病原体为对虾皮下?..     

Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection

An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue–green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140 nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0 kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e−124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV.     

犬传染性肝炎病毒在体外细胞质内的发生

通过对犬传染性肝炎病毒（ＩＣＨＶ）在犬肾传代细胞内形态发生及其抗原定位的电镜和免疫胶体金电镜研究,发现ＩＣＨＶ除了在宿主细胞核内发生外,还有一条细胞质内的发生途径。在细胞质内病毒核壳体的装配是以均质致密包涵体和副晶格包涵体为“基地”,这与人们熟知的细胞核内形态发生方式相似。免疫胶体金标记显示,细胞质包涵体中含有大量的ＩＣＨＶ抗原成分,显核壳体在细胞质内装配病毒的结构蛋白来源。此外,在感染的细胞质内还观察到与核内相同的病毒核心样结构。     

Subacute sclerosing panencephalitis (SSPE): isolation of a defective variant of measles virus from brain obtained at autopsy.

A cytopathic agent causing formation of syncytial giant cells was isolated by co-cultivation of human embryonic lung cells with brain cells obtained at autopsy from a patient with subacute sclerosing panencephalitis. Measles specific intracellular immunofluorescence was detected in syncytial giant cells developed by the agent. Paramyxovirus-like nucleocapsids were observed by electron microscopy in nuclei of the syncytial giant cells. Measles specific immunofluorescence was also detected on the surface of unfixed syncytial giant cells. However, the synycytial giant cells did not produce either virions or hemogglutinin, and did not show hemadsorption of African green monkey red cells. Hence, the newly isolated agent seems to be a defective variant of measles virus, and was designated as the SSPE-"BIKEN" strain.     

Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei

White spot syndrome virus (WSSV) causes disease and mortality in cultured and wild shrimp. A standardized WSSV oral inoculation procedure was used in specific pathogen-free (SPF) Litopenaeus vannamei (also called Penaeus vannamei) to determine the primary sites of replication (portal of entry), to analyze the viral spread and to propose the cause of death. Shrimp were inoculated orally with a low (10(1.5) shrimp infectious dose 50% endpoint [SID50]) or a high (10(4) SID50) dose. Per dose, 6 shrimp were collected at 0, 6, 12, 18, 24, 36, 48 and 60 h post inoculation (hpi). WSSV-infected cells were located in tissues by immunohistochemistry and in hemolymph by indirect immunofluorescence. Cell-free hemolymph was examined for WSSV DNA using 1-step PCR. Tissues and cell-free hemolymph were first positive at 18 hpi (low dose) or at 12 hpi (high dose). With the 2 doses, primary replication was found in cells of the foregut and gills. The antennal gland was an additional primary replication site at the high dose. WSSV-infected cells were found in the hemolymph starting from 36 hpi. At 60 hpi, the percentage of WSSV-infected cells was 36 for the epithelial cells of the foregut and 27 for the epithelial cells of the integument; the number of WSSV-infected cells per mm2 was 98 for the gills, 26 for the antennal gland, 78 for the hematopoietic tissue and 49 for the lymphoid organ. Areas of necrosis were observed in infected tissues starting from 48 hpi (low dose) or 36 hpi (high dose). Since the foregut, gills, antennal gland and integument are essential for the maintenance of shrimp homeostasis, it is likely that WSSV infection leads to death due to their dysfunction.     

Observation by electron microscopy of a gregarine parasite of Antarctic krill: its histological aspects and ecological explanations

Antarctic krill are parasitized by gregarines (Phylum Apicomplexa, Class Sporozoea, Order Eugregarinida), which were observed in this study by light, scanning, and transmission electron microscopy. Eighty seven percentage of the krill examined (n = 93) were infected with the parasites in the cephalin stage, which were regularly found in the hind-gut epithelium of their host. Cephalins were found attached to the epithelium, and then were liberated into the intestinal lumen. The gamont stage, which follows the cephalin stage, was found in the intestinal lumen, as well as the diverticulum of mid-gut gland. However, gamonts found in the mid-gut gland were considerably larger and elongated in comparison with those in the intestinal lumen. Gamonts in the diverticulum appear to damage microvilli, which are involved in the uptake of digested nutrients and secretion of various enzymes. Therefore, elevated infestation of this parasite in the mid-gut gland may have a significant impact on the nutritional state of the Antarctic krill host.     

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.     

A new virus associated with the parasitoid Cotesia marginiventris (Hymenoptera: Braconidae): Replication in noctuid host larvae

A nonoccluded filamentous baculo-like virus occurred in scattered hypodermal and tracheal matrix cells of five species of noctuid larvae (Spodoptera frugiperda, S. exigua, S. ornithogalli, Heliothis zea, and Trichoplusia ni) parasitized by Cotesia (= Apanteles) marginiventris. Infected cells and their nuclei were moderately hypertrophied. Infected cells contained prominent virogenic stromata, fibrous cytoplasmic inclusions, and moderate numbers of a filamentous virus. Virions were 50–98 nm in diameter, ≥865 nm in length, and straight to slightly flexuous. Nucleocapsids were 30–39 nm in diameter with a core 21–24 nm in diameter, and were enclosed by one or two envelopes. Nucleocapsids occurred in the nuclei and in the cytoplasm. Enveloped nucleocapsids were restricted to the cytoplasm.