Ultrastructural study of poxvirus causing myxomatosis in rabbits, in São Paulo and Santa Catarina, Brazil / Estudio ultraestructural del poxvirus causal de mixomatosis en conejos, en São Paulo y Santa Catarina, Brasil

The myxomatosis is a contagious worldwide disease caused by poxvirus which infects domestic and wild rabbits. In the present study we present two distinct outbreaks of myxomatosis when raising rabbits, one for commercial purpose of production of meat and skins and, another one for the commercialization of ornamental rabbits. The observed signs were ocular, auricular, nasal, testis lesions and many times scattered throughout the body of the animals. The lesions were characterized by formation of nodules that by palpation disclosed gummy or gelatinous aspect. At the transmission electron microscopy, all the skin and crust samples were analyzed by negative staining technique. A great number of particles with morphology similar to the poxvirus, some enveloped in a brick-shaped and irregular disposition of tubules on the external membrane, measuring 300x240 nm on the average were visualized. Ultra thin sections revealed the presence of intracytoplasmic inclusion bodies surrounded by membrane containing oval particles, measuring 270 x 130 nm, containing nucleus or an internal biconcave (dumbbell-shaped) core. Immature particles (empty), surrounded by membrane were also observed. In addition, intracytoplasmic electron dense inclusion bodies containing viral particles budding of dense amorphous material and intranuclear fibrillar or "digital" inclusions showing a regular striation and arranged in groups were found in the middle of granular material. The nuclei were deformed with densely condensed chromatin forming amorphous and electron dense inclusion bodies. In the immunocytochemistry technique, the antigen-antibody reaction was strongly marked by the particles of colloidal gold, emphasizing the viral particles. The techniques used in this study were important in the diagnosis of the affected animals.

La mixomatosis es una enfermedad contagiosa de distribución mundial, causada por poxvirus que infecta conejos domésticos y salvajes. En este estudio presentamos dos distintos surtos por mixomatosis que ocurrieron en producciones de conejos, una para fines comerciales de producción de carne y pieles y otra para el comercio de conejos domésticos. Las señales observadas fueron afecciones oculares, nasales, testiculares y, a veces, también distribuida por todo el cuerpo de los animales. Estas se caracterizaban por formación de nódulos que a la palpación tenían un aspecto gelatinoso o gomoso. En la microscopía electrónica de transmisión, por la técnica de contrastación negativa, se pudo observar en todas las muestras examinadas de piel y de costras, un gran número de partículas típicas de poxvirus, con envoltura y forma de ladrillo, mostrando disposición irregular de los túbulos sobre la membrana externa, midiendo 300 x 240 nm en el promedio. Cortes ultrafinos de fragmentos de piel y de costras revelaron la presencia de cuerpos de inclusión intracitoplasmáticas, envueltos por membrana y conteniendo partículas ovales, midiendo 270 x 130 nm, conteniendo núcleo o centro interno bicóncavo (forma de mancuernas). Partículas inmaduras (vacías) envueltas por membrana fueron observadas. También fueron analizados cuerpos de inclusión intracitoplasmáticos, electrodensos, conteniendo partículas virales brotando del material denso y amorfo. Fueron observadas inclusiones intranucleares fibrilares o "digitales" mostrando una estriación periódica y disposición en grupos en medio del material granular. Los núcleos estaban deformados con cromatina densamente condensada formando cuerpos de inclusiones electrodensas y amorfas. En la técnica de imunocitoquímica la reacción antígeno-anticuerpo fue intensamente marcada por las partículas de oro coloidal realzando fuertemente las partículas virales.

Ultrastructural study of myxoma virus morphogenesis.

Poxviruses are among the largest and most complex viruses known. Vaccinia virus, the prototype of the family Poxviridae, has been studied much more than myxoma virus. The aim of this work was to have a better knowledge about myxoma virus morphogenesis. The characterization of the main stages of MV morphogenesis was achieved by ultrastructural and immunological analysis. Specific antibodies were raised against M022L and M071L, two envelope proteins of extracellular enveloped virus and intracellular mature virus, respectively. The main stages of assembly were similar to those seen with other poxviruses, and the duration of the whole replication cycle was estimated to be around 16 h, longer than what was described for vaccinia virus. Morphological changes of infected cells were associated with the development of long cellular projections and enlarged microvilli. Intracellular enveloped viruses are associated with the cytoskeleton to move through the cell. Unlike earlier studies, as many cell-associated enveloped viruses as intracellular enveloped viruses were observed in relation with specialized microvilli, although these structures were rarely noticed. Finally, an unusual spreading process was observed, which uses cytoplasmic corridors.

Ligand-induced and nonfusogenic dissolution of a viral membrane.

Hitherto, all enveloped viruses were thought to shed their lipid membrane during entry into cells by membrane fusion. The extracellular form of Vaccinia virus has two lipid envelopes surrounding the virus core, and consequently a single fusion event will not deliver a naked core into the cell. Here we report a previously underscribed mechanism in which the outer viral membrane is disrupted by a ligand-induced nonfusogenic reaction, followed by the fusion of the inner viral membrane with the plasma membrane and penetration of the virus core into the cytoplasm. The dissolution of the outer envelope depends on interactions with cellular polyanionic molecules and requires the virus glycoproteins A34 and B5. This discovery represents a remarkable example of how viruses manipulate biological membranes, solves the topological problem of how a double-enveloped virus enters cells, reveals a new effect of polyanions on viruses, and provides a therapeutic approach for treatment of poxvirus infections, such as smallpox.

Occurrence of atypical myxomatosis in Central Europe: clinical and virological examinations.

An outbreak of the atypical form of myxomatosis struck a rabbit farm in Hungary. The animals had previously been vaccinated with a vaccine containing Shope rabbit fibroma virus strain. The disease appeared in winter when the presence of mosquitoes and fleas is not common. The virus was isolated from an eyelid specimen of a naturally infected rabbit. The surviving animals were observed for four weeks, blood samples were collected and, after euthanasia, organ specimens were also examined by morphological methods including pathology and electron microscopy. Serum samples were examined by virus neutralisation for antibodies. Genetic analysis of the isolated virus was carried out by polymerase chain reaction (PCR) and direct sequencing. The primers were designed on the basis of the major envelope gene (Env) of the Lausanne reference strain in the GenBank. The viral proteins were examined by SDS-PAGE. The isolated virus (ref. no.: BP04/2001) was able to infect the susceptible animals directly, by contact. The disease was characterised by respiratory symptoms of the upper tracheal tract, conjunctivitis and high mortality by the 11th-14th day. Aerogenic infection with strain BP04/2001 resulted in 100% morbidity among the susceptible animals. Sequencing of the amplified 400-bp-long DNA revealed 97% homology with the Env gene of the Lausanne strain, which proves that strain BP04/2001 is a variant of the Lausanne strain having been enzootic throughout Europe. The live vaccine strain used in Hungary against myxomatosis, which is also a Lausanne-derived strain, protected the animals. According to the protein analysis a protein of 200 kDa in size is not expressed in strain BP04/2001. This is the first report on atypical myxomatosis in Central Europe. The virus spreads by airborne transmission and may cause severe losses in the rabbit population.

Isolation and plaque purification of a syncytial forming component of myxoma virus.

Using the endpoint dilution and plaque purification technique performed on rabbit kidney cells, two stable syncytial clones of myxoma virus have been isolated. No eosinophilic intracytoplasmic inclusion bodies nor intranuclear granulations could be detected in cells infected with these clones. Traditional plaques with the central area of detached cells surrounded by round cells also could not be recognized. However, when enough virus was added, one large complete syncytium developed in 24 on the monolayer. Syncytial activity was associated with the entire particle. This was the first time that myxoma virus is reported to demonstrate such an intense syncytial activity.

Malignant rabbit fibroma virus: observations on the culture and histopathologic characteristics of a new virus-induced rabbit tumor.

The clinical, histopathologic, and cultural characteristics of a newly isolated poxvirus, malignant rabbit fibroma virus (MV), were investigated. MV was isolated from tumors induced by an uncloned stock of Shope fibroma virus (SFV). MV, SFV, and rabbit myxoma virus were compared. Similarly to myxoma virus, MV grew to higher titer in vitro than did SFV and produced plaques rather than foci on rabbit kidney cell monolayers. Unlike the local, self-limited fibroblastic proliferations observed in SFV recipients, MV and myxoma caused a fulminant clinical syndrome characterized by malignant histology, metastases, and supervening fatal gram-negative infection with Pasteurella multocida. MV induced a large, protuberant local tumor and discrete metastases histologically resembling myxosarcomas. Draining lymph nodes contained metastases and showed diffuse cortical hyperplasia. Kupffer's cells were prominent in the liver, and macrophages were abundant in the splenic sinusoids. The lungs and trachea were spared, but the conjunctiva and nasal mucosa showed squamous metaplasia and atypia, with overlying Pasteurella infection and underlying tumor. Myxoma virus infection produced similar mucosal changes, but both of these as well as the epidermis overlying the myxomas showed cytoplasmic virus inclusions. Neither the skin nor the epithelial surfaces overlying MV-induced tumors nor the tumors themselves contained virus inclusions. Thus the tumor syndrome caused by MV differed from other known rabbit tumors. Endonuclease restriction digests showed that the MV genome resembled, but was distinct from, rabbit myxoma virus. Opportunistic infection associated with MV-induced disseminated tumor may be an experimental model for the infectious complications that often supervene in host-tumor relationships.