Characterization of infectious laryngotracheitis viruses, antigenic comparison by kinetics of neutralization and immunization studies.

Five isolates of infectious laryngotracheitis virus were compared by pock formation on the chorioallantoic membrane of embryonated eggs, plaque size in chicken embryo kidney tissue culture, and antigenic relationship using reciprocal kinetics of neutralization. The A4557-5 strain of infectious laryngotracheitis virus, which causes mild respiratory disease, produced pocks with a zone of edema on the chorioallantoic membrane. A virulent virus (Virus 1), isolated from an outbreak of severe disease characterized by a diphtheritic laryngotracheitis, produced the largest plaques in chicken embryo kidney cell culture. Other virulent viruses (Viruses 2, 3 and V154) did not have unique growth characteristics when grown on the chorioallantoic membrane or in chicken embryo kidney cell culture. All viruses were closely related antigenically as shown by kinetics of neutralization but viruses 2 and 3 were not homogeneous with the other three viruses when neutralized by anti-V154 chicken serum. Following aerosol infection, chickens infected with the A4557-5 virus were immune to challenge with virulent V154 virus. However, in comparison to SA-2 virus, this virus was a less effective immunizing agent when administered by the vent or drinking water methods.     

Immuno-gold labelling of turkey rhinotracheitis virus.

The morphology and morphogenesis of five viruses isolated in Great Britain, France and South Africa from turkeys with rhinotracheitis were examined. The five isolates were antigenically related by immunofluorescence and were indistinguishable by negative contrast and thin section electron microscopy. Negative contrast electron microscopy of infected Vero cell cultures revealed ortho- or paramyxovirus-like particles and helical nucleocapsids 14 nm in diameter with a pitch of 6 nm. The viral nature of these structures was confirmed by immuno-gold labelling, using a hyperimmune rabbit antiserum to TRT virus and 15 nm gold-labelled goat anti-rabbit IgG. Ultrastructural changes characteristics of paramyxovirus infection were observed in Vero cell cultures infected with each of the five TRT virus isolates. These alterations, which included areas of localised thickening of plasma membrane, associated cytoplasmic inclusions of nucleocapsids and budding virus particles also labelled specifically following immunogold staining. These observations are in accord with the suggestion that TRT virus is an avian pneumovirus.     

Studies with an unclassified virus isolated from diarrheic calves

A transmissible agent (Breda agent) was isolated from a calf with diarrhea and shown to be infectious by inoculation orally into gnotobiotic and conventionally reared calves. The “Breda” agent had the morphology of a virus and possessed a hemagglutinin. Antigenic studies showed the virus to be antigenically different from bovine coronavirus, parainfluenza 3 virus, bovine rotavirus, bovine parvovirus and bovine pestivirus (BVD). Attempts to culture the virus in cell or organ cultures or in embryonated eggs, were unsuccessful. The virus was either spherical or kidney shaped, with 7–9 nm peplomers on the surface. A few particles possessed coronavirus processes of 17–20 nm, but these were arranged irregularly and were thought to be tissue debris. Three out of eight experimental calves developed severe diarrhea and the lesions in the small and large intestines were similar to those reported for coronavirus. The virus replicated in the jejunal and ileal regions of the small intestine and in the spiral colon, as judged by immunofluorescence. The virus multiplied in all experimental calves and was excreted in the feces; excretion correlating with the onset of diarrhea or a change in the appearance of the feces. There was little or no malabsorption measured by the uptake of D-xylose and the fact that infection of both the crypt and villus epithelial cells was observed, suggests that the pathogenesis may be different from rotavirus and coronavirus. Fourteen of fortyseven calves in the outbreak were infected with the virus, virus was not identified in other farm outbreaks of the disease.     

Sensitivity and specificity of the fluorescent antibody technique for detection of infectious laryngotracheitis virus.

The specificity of a fluorescent conjugate to infectious laryngotracheitis virus was examined using chick trachea organ culture or tissue sections infected with other avian viruses (adenovirus, infectious bronchitis, poxvirus, reovirus, Newcastle disease virus, Marek's disease virus, avian encephalomyelitis and infectious bursal agent) or Mycoplasma gallisepticum. Confirmation of virus replication in these preparations was obtained by either 1) demonstration of virus titre increase or 2) demonstration of fluorescence when using the homologous conjugate. Once either of these criteria had been satisfied, negative results with the infectious laryngotracheitis conjugate were taken to indicate that the conjugate would not present false positive results in differentiated cells infected with these heterologous viruses. The spectrum of reactivity of the infectious laryngotracheitis conjugate was then examined on organ cultures infected with several infectious laryngotracheitis isolates from across Canada. Finally, the conjugate was applied to experimental and natural cases of infectious laryngotracheitis and its efficiency was compared to routine virus isolation methods.     

Isolation of visna-maedi virus from the choroid plexus of an apparently healthy sheep in Italy

In this paper, the presence of visna-maedi in Italy, reported to exist since 1982 on clinical grounds, and the presence of anti-visna-maedi antibody is confirmed by the isolation of the viral agent from choroid plexus cells of an apparently healthy sheep. Gel diffusion test antigen, prepared from the explanted choroid plexus cells and from normal choroid plexus cells infected with the isolated agent, gave a positive reaction with one or two identity lines with reference antigens. The isolated agent gave both a characteristic cytopathic effect and a cytoplasmic fluorescence starting 24 hours after infection in normal choroid plexus cell cultures. Fluorescence was observed neither in control normal choroid plexus cells nor in infected normal choroid plexus cells incubated with a visna-maedi negative serum. By electron microscopy, budding forms arising from the cell membrane and extracellular particles of two distinct types were observed. One type was characterized by an electron-dense central core and a single membrane, and ranged from 80 to 110 nm, while the other had elongated bar-like cores and a double wall, and ranged from 100 to 140 nm. The characteristics observed for the isolated agent are identical to those reported by various authors for visna-maedi virus.     

Persistent infection of bovine herpesvirus type 4 in bovine endothelial cell cultures

Herpesviruses can establish a persistent infection in the cells and tissues of their natural hosts and thus may produce diseases due to cytolytic infections. We have isolated a herpesvirus from a bovine vascular endothelial cell culture after continuous subculturing. Typical cytopathic changes were observed in bovine endothelial cell cultures 2 days after inoculation of the virus. The virus had an icosahedral nucleocapsid of 100-150 nm in diameter and an envelope. The sequences of some DNA fragments of the virus were highly homologous to those of the bovine herpesvirus type 4 (BHV-4) strains. The DNA restriction maps of the virus and the reference strains of BHV-4, DN 599 and Movar 33/63 were very similar but not identical. Therefore, the newly isolated virus has been designated Taiwan strain. The presence of BHV-4 DNA in apparently normal bovine endothelial cell cultures was shown by Southern blot hybridization with the BamHI fragment of the newly isolated BHV-4 and was further confirmed by digestion of the DNA with BamHI plus AccI. In conclusion, we have demonstrated that BHV-4 persisted in the bovine endothelial cell cultures and continuous subcultures could lead to the production of infectious viral particles.     

Detection of cell membrane proteins that interact with virulent infectious bursal disease virus

To detect the molecules that interact with infectious bursal disease virus (IBDV), the chicken B lymphoblastoid cell line, LSCC-BK3, which is permissive for virulent IBDV infection was investigated. The sodium dodecyl sulfate-solubilized plasma membrane fraction from the cells was subjected to a virus overlay protein binding assay. The IBDV specifically bound to proteins in LSCC-BK3 plasma membranes with molecular weights of 70, 82 and 110 kDa. This is the first report to demonstrate cellular molecules that interact with virulent IBDV.     

The propagation of avian viruses in a continuous cell line (QT35) of Japanese quail origin

Seven of nine avian virus families tested (Birnaviridae, Coronaviridae, Herpesviridae, Paramyxoviridae, Poxviridae, Reoviridae, and Retroviridae) were found to replicate in a quail fibroblast cell line, designated QT35, resulting in a cytopathic effect (CPE) visible with the naked eye or by low-power microscopy. In comparison, only one (Paramyxoviridae) of seven mammalian virus families tested produced an observable CPE. Cytopathic changes induced by examined viruses were round cell, syncytial, and focus formation. Trypsin did not promote cytopathic changes by selected CPE-negative avian and mammalian viruses in QT35 cells. Several avian viruses (infectious bursal disease virus, Newcastle disease virus, Canary pox virus, and reovirus) formed plaques under agar. Avian reovirus and infectious bursal disease virus produced similar titers in chicken embryo fibroblast (CEF) and QT35 cell cultures. Chicken-egg-yolk neutralizing-antibody titers to IBDV were comparable in CEF and QT35 cell-culture systems.     

Isolation of visna-maedi virus from the choroid plexus of an apparently healthy sheep in Italy

In this paper, the presence of visna-maedi in Italy, reported to exist since 1982 on clinical grounds, and the presence of anti-visna-maedi antibody is confirmed by the isolation of the viral agent from choroid plexus cells of an apparently healthy sheep.Gel diffusion test antigen, prepared from the explanted choroid plexus cells and from normal choroid plexus cells infected with the isolated agent, gave a positive reaction with one or two identity lines with reference antigens. The isolated agent gave both a characteristic cytopathic effect and a cytoplasmic fluorescence starting 24 hours after infection in normal choroid plexus cell cultures.Fluorescence was observed neither in control normal choroid plexus cells nor in infected normal choroid plexus cells incubated with a visna-maedi negative serum.By electron microscopy, budding forms arising from the cell membrane and extracellular particles of two distinct types were observed. One type was characterized by an electron-dense central core and a single membrane, and ranged from 80 to 110 nm, while the other had elongated bar-like cores and a double wall, and ranged from 100 to 140 nm. The characteristics observed for the isolated agent are identical to those reported by various authors for visna-maedi virus.     

THE ISOLATION OF LENTOGENIC STRAINS OF NEWCASTLE DISEASE VIRUS IN AUSTRALIA

SUMMARY Twelve isolations of Newcastle disease virus were made from 77 clinical samples from chickens with conjunctivitis, respiratory disease, proventriculitis and bursal atrophy. Nine of the Isolations were made from chickens with conjunctivitis. The viruses were identified as Newcastle disease virus by inhibition of their haemagglutinins with specific antiserum to Newcastle disease virus. The viruses failed to kill chicken embryos after inoculation into the allantoic cavity and they were judged to be lentogenic strains. There was no evidence that the Newcastle disease viruses were responsible for any of the clinical conditions from which they were isolated. The presence of other agents in 10 of the samples was indicated by reduced production of haemagglutinin in allantoic fluids of infected embryos, by deaths of infected embryos, by the production of cytopathic changes in avian cell cultures and by electron microscopy. Three isolations of infectious bronchitis virus, 2 of avian adenovirus and one of avian reovirus were made. Other samples were suspected of containing infectious bronchitis virus and mycoplasmas, but these were not isolated. The Newcastle disease viruses failed to produce plaques in chicken embryo fibroblast cell cultures and they were separated from the contaminating agents by haemagglutination and elution followed by passage at terminal dilution in chick embryos. No Newcastle disease virus was isolated from 60 caecal tonsils and 60 lung samples from 9-week-old broiler chickens. Eight lung samples yielded mycoplasmas that caused haemadsorption in chicken cell cultures. The mycoplasmas were probably Mycoplasma gallisepticum.     

Infectious Bursal Disease in New Brunswick

A flock of four week old chickens experienced a disease of sudden onset in which the only symptoms were those of depression shortly before death, and in which the predominant histological lesion was necrosis of lymphocytes in the bursa of Fabricius.

A virus, designated strain Sk-1, was isolated from pooled bursal tissue of affected birds and was serologically identified as a strain of the infectious bursal agent. This virus was chloroform resistant, did not hemagglutinate guinea pig or chicken erythrocytes and did not produce a cytopathic effect in chick embryo tissue cultures. Equivocal results were obtained in filtration studies but the agent was less than 100nm in diameter.

Four week old chicks inoculated with strain Sk-1 developed microscopic lesions in the bursa of Fabricius which were similar to those seen in the original field specimens. Inoculated chick embryos exhibited characteristic macroscopic lesions and necrosis of vascular tissue was a common histological change.

A limited serological survey of local poultry flocks indicated that infection by this agent had occurred in four of the ten flocks examined.

     

Virus-like particles, 45 to 65 nm, in intestinal contents of neonatal calves.

Enveloped virus particles 45 to 65 nm in diameter, tentatively called minicorona virus, were detected by electron microscopy in the intestinal contents of one normal and seven diarrheic calves in Quebec dairy herds. The agent was shown to be antigenically unrelated to the Nebraska calf diarrhea coronavirus and to the bovine viral diarrhea virus by counterimmunoelectrophoresis and fluorescent-antibody techniques. Antibodies against these particles were demonstrated in the serum of affected calves using immunoelectron microscopy. The agent could not be isolated in cell cultures and its possible role as etiological agent in calf diarrhea is still to be determined.     

Morphologic and physical characteristics of feline infectious peritonitis virus and its growth in autochthonous peritoneal cell cultures.

Characteristic viral-type particles were seen in liver of kittens experimentally infected with the feline infectious peritonitis (FIP) agent. The particles were from 70 to 75 nm in diameter, with a central doughnut-shaped nucleoid 50 to 55 nm in diameter; numerous spikelike projections extended from their envelopes. Similar particles were seen by electron microscopy in peritoneal cell cultures derived from the peritoneal exudate of experimentally infected kittens, and viral antigens were identified in these cells by immunofluorescence. Cells and supernatant fluids from cultures containing these particles produced FIP when injected into the peritoneal cavity of kittens. The FIP agent is heat sensitive, ether labile, and relatively phenol resistant and is inactivated within 24 hours at room temperature. The FIP agent is inactivated by recommended viricidal concentrations of chlorhexidine and benzlkonium chloride.     

新城疫病毒囊膜糖蛋白生物学特性的研究进展

新城疫是当今全球范围内最严重的禽类传染病之一。其病原新城疫病毒是单股负链RNA病毒,编码NP、P、M、F、HN和L 6种结构蛋白,其中最主要的是位于囊膜上的F和HN两种糖蛋白。F蛋白具有使病毒囊膜与宿主细胞膜融合,致使病毒穿入宿主细胞膜的作用,是决定病毒毒力的关键因子;HN蛋白具有血凝素和神经氨酸酶两种活性,这两种活性对于NDV侵染细胞具有重要的作用。这两种糖蛋白不仅对NDV的毒力及致病性方面起着决定性作用,而且也是诱导产生保护性抗体的蛋白。作者主要对这两种蛋白的结构与功能作一概述。     

The development of a purification method for Borna disease virus

Borna disease virus represents an unknown neurotropic agent. It causes encephalitis in horses and sheep. The same or a similar type of virus might be responsible for psychiatric disorders in man. So far, it has been impossible to purify this agent to such an extent that it could be analyzed biochemically or electronmicroscopically. Therefore, different conventional virus purification techniques are applied in order to develop a method for obtaining purified Borna disease virus from infectious rat brain or persistently infected cell culture material.     

Persistent infections of a field strain of rabies virus in murine neuroblastoma (NA-C1300) cell cultures.

Rabies virus from the brain of a striped skunk (Mephitis mephitis) from Ontario was inoculated into murine neuroblastoma (NA-C1300) cell cultures. These cultures were incubated and the cells were subcultured every three to four days. The presence of viral antigen in the cell cultures was monitored by direct immunofluorescent staining and in the culture fluids by titration in either baby hamster kidney (BHK/C13) or NA cells or in experimental mice. The virus-infected NA cultures evolved from an initial high viral concentration in supernatant fluid through a period of decreasing titers of infectious virus in the supernatant fluids to a final phase where no infectious virus has been found following cell culture and animal inoculation methods attempted although the persistently infected cells remained 95-100% viral nucleocapsid antigen-positive. Possible mechanisms involved in the perpetuation of this infection are discussed. This is the first report of a persistent infection of cell cultures by a field strain of rabies virus.     

Chicken infectious anemia in Mexico: virus identification and serology survey.

Three chicken infectious anemia (CIA) virus strains were isolated from 10 different sick broiler and replacement chicken flocks with the MDCC-MSB1 cell line. One-day-old specific-pathogen-free chicks were inoculated later, with the three original samples being positive in tissue culture; one induced signs and lesions, another only lesions typical for CIA. One isolate was selected for further trials and showed resistance to chloroform and heat (75 C for 5 min) and passed through a 45-nm filter membrane but did not pass through the 22-nm filter. These characteristics were similar to the Del Rose reference strain of chicken anemia virus. By electron microscopy, the diameter of particles obtained from the pellet of infected cell cultures was between 22 and 27 nm. Serology survey carried out with 580 serum samples from different poultry farms all over the country with a commercial enzyme-linked immunosorbent assay kit gave proof of widespread seroconversion, indicating that CIA should be considered endemic to Mexico.     

鸡传染性支气管炎病毒受体研究进展

鸡传染性支气管炎是一种世界性分布的严重影响养鸡业的高度接触性传染病,其病原是一种最早发现的冠状病毒鸡传染性支气管炎病毒(IBV)。IBV入侵细胞的受体是目前IBV研究中的热点之一。已知IBV囊膜上的纤突蛋白(S)是宿主组织和细胞嗜性及致病性的主要决定因素,S蛋白通常断裂为S1和S2两部分,S1与宿主细胞膜上的细胞受体相结合,在S2的作用下病毒囊膜与宿主细胞膜融合,IBV与受体结合之后于中性或微碱性pH时发生构象改变,从而获得病毒进入宿主细胞的能力。对于IBV的受体研究主要集中于IBV可能使用的几种功能性受体,包括氨肽酶N、唾液酸、硫酸乙酰肝素等。     

Virus Pneumonia of Pigs; Attempts at Propagation of the Causative Agent in Cell Cultures and Chicken Embryos

Two strains of the agent of virus pneumonia, were tested for the ability to propagate in 12 types of cell cultures and in chicken embryos. The 5 primary cell cultures used were: swine kidney, lung, bone marrow, testicle, and chicken embryo kidney; and the 7 serial passage cell cultures were: swine kidney, kidney-tumor, testicle, bone-marrow, bovine kidney, and human cervical carcinoma (HeLa). The agent of virus pneumonia was propagated in primary swine kidney and in HeLa cell cultures as shown by the production of typical gross and microscopic lesions in pigs inoculated with cell future fluids. Third passage cell culture fluids, produced typical gross lesions in pigs, but fourth passage cell culture fluids produced only microscopic lesions, and no lesions were produced by sixth and eleventh passage fluids. Control pigs receiving fluids from uninoculated cell cultures remained free of gross or microscopic lesions, as did uninoculated controls. Cytopathic effects were not detected in any of the inoculated cell cultures and no cellular changes were detected by staining with Giemsa stain or acridine orange.

Neither lesions nor deaths occurred in chicken embryos inoculated with both strains of virus pneumonia virus. Pneumonia was not produced in pigs inoculated with suspensions from second chicken embryo passage of the 2 strains inoculated by the chorioallantioic sac, the amniotic sac, and the yolk sac routes.

Identical gross and microscopic lesions were produced in pigs inoculated with either pneumonic lung suspensions or with virulent cell culture fluids. Gross lesions consisted of areas of light to reddish-purple consolidation usually limited to the anterior, cardiac, and intermediate lobes of the lungs. Pleuritis and pericarditis were never present in experimentally produced virus pneumonia. The microscopic lesions were characterized by: 1. perivascular and peribronchiolar lymphoid infiltration and hyperplasia, 2. alveolar interstitial thickening and infiltration, and 3. alveolar exudates consisting of alveolar cells, lymphocytes, plasma cells, and neutrophiles.

     

Detection of African malignant catarrhal fever virus antigens in cell cultures by immunofluorescence

A fluorescent antibody (FA) test for antigens of African bovine wildebeest-derived malignant catarrhal fever virus was developed. Serum from one of the few survivors of the experimental disease in steers was used to prepare the conjugate. Both a virulent and an attenuated strain of malignant catarrhal fever virus were used to infect bovine thyroid cell cultures. Cells infected with both strains were readily detected by FA staining as early as 24 h post infection, whereas cytopathic effect could be observed by bright-field microscopy only after days 5 or 6 post infection. Controls consisting of normal bovine thyroid cells or infected cells treated with conjugated normal globulins did not show autofluorescence. The reaction was blocked by treatment of infected cells with homologous positive antisera but not by treatment with normal bovine serum or antisera to foot-and-mouth disease, rinderpest, bovine virus diarrhea, Ibaraki, infectious bovine rhinotracheitis, or bovine herpes mammilitis viruses. Treated with African malgnant catarrhal fever virus conjugate did not react.