Chorioallantoic Membrane Of Embryonated Eggs Research Articles

Isolation, identification, molecular and pathogenicity characteristics of an infectious laryngotracheitis virus from Hubei province, China

Multiple outbreaks of avian infectious laryngotracheitis (ILT) in chickens, both domestically and internationally, have been directly correlate to widespread vaccine use in affected countries and regions. Phylogenetic and recombination event analyses have demonstrated that avian infectious laryngotracheitis virus (ILTV) field strains are progressively evolving toward the chicken embryo-origin (CEO) vaccine strain. Even with standardized biosecurity measures and effective prevention and control strategies implemented on large-scale farms, continuous ILT outbreaks result in significant economic losses to the poultry industry worldwide. These outbreaks undoubtedly hinder efforts to control and eradicate ILTV in the future. In this study, an ILTV isolate was successfully obtained by laboratory PCR detection and virus isolation from chickens that exhibited dyspnea and depression on a broiler farm in Hubei Province, China. The isolated strain exhibited robust propagation on chorioallantoic membranes of embryonated eggs, but failed to establish effective infection in chicken hepatocellular carcinoma (LMH) cells. Phylogenetic analysis revealed a unique T441P point mutation in the gJ protein of the isolate. Animal experiments confirmed the virulence of this strain, as it induced mortality in 6-wk-old chickens. This study expands current understanding of the epidemiology, genetic variations, and pathogenicity of ILTV isolates circulating domestically, contributing to the elucidate of ILTV molecular basis of pathogenicity and development of vaccine.

Co-delivery of beta-caryophyllene and indomethacin in the oily core of nanoemulsions potentiates the anti-inflammatory effect in LPS-stimulated macrophage model

The potentiation of pharmacological effects can be achieved through several strategies, such as the association of substances and delivery in nanostructured systems. In practice, potentiation can be measured by the law of mass action and joint evaluation of the combination index (CI) and dose–response curves. In this context, this study aimed to evaluate the anti-inflammatory effect of the association of β-caryophyllene and indomethacin in the free form and delivered in nanoemulsions using the in vitro model of LPS-stimulated murine macrophage. The results indicated potentiation of the anti-inflammatory effect of nanoemulsified substances compared to free substances, as well as synergistic action between the sesquiterpene and the selected NSAID. In comparison, the association of β-caryophyllene and indomethacin in the free form inhibited the production of nitric oxide by 50% at 48.60 µg/mL (CI = 0.21), while the nanoemulsified association of these substances resulted in an IC50 of 1.45 µg/mL (CI = 0.14). In parallel, cytotoxicity assays on HaCaT and MRC-5 cell lines demonstrated the safety of IC50-equivalent concentrations of the anti-inflammatory action, and no irritating effects on the chorioallantoic membrane of embryonated eggs were observed (HET-CAM assay). The results suggest that β-caryophyllene may be an alternative to replace an inert oily core in nanoemulsion systems when anti-inflammatory effects are desirable.

Isolation and characterization of avipoxviruses from wild birds in Western Australia.

Avipoxviruses were isolated from wart-like lesions in an Australian magpie (Gymnorhina tibicen) and a silvereye (Zosterops lateralis), and the poxvirus aetiology of wart-like lesions in a magpie-lark (Grallina cyanoleuca) was confirmed. The three viruses produced typical pock lesions on the chorioallantoic membrane of embryonated eggs and were able to replicate in trypsin-dispersed chick embryo fibroblast cultures but not confluent monolayer cultures. Pock neutralization and immunodiffusion studies showed that the three wild bird isolates were distinct from fowlpox, although antigenically closer to fowlpox than pigeonpox. The magpie and silvereye isolates were more closely related to each other than to the magpie-lark isolate.

An egg-adapted (attenuated) line of Eimeria necatrix: further studies on its reproduction, pathogenicity and immunogenicity.

An egg-adapted line of Eimeria necatrix has now been passaged 40 times in the chorio-allantoic membranes of embryonated eggs. Between the 21st and 40th passage in eggs the parasite was subjected to a selection for precocious development, and its pre-patent period in this host is now 123-125 h. The parasite continued to adapt to the chorio-allantoic membrane throughout the period of the passages and its development in this tissue caused the growth of the host embryo to be markedly stunted. In chickens, both the reproduction and the pathogenicity of the egg-adapted line decreased with increasing numbers of passages in eggs. A comparison of the immunogenicity of the egg-adapted line and its parent strain in chickens kept on litter, showed that substantially more oocysts of the egg-adapted line had to be given to induce complete protection.

Effect of betamethasone-17-valerate on synthesis of collagen and prolyl hydroxylase activity in chick embryo tendon cells

The effect of betamethasone-17-valerate on the biosynthesis of collagen was studied in matrix-free chick embryo tendon cells in vitro and the activity of prolyl hydroxylase was assayed in the cells after incubation with the steroid in vitro and after injection of the steroid on to chorioallantoic membrane of embryonated eggs. Tendon cells synthesized collagen at a rapid rate and the synthesis was essentially linear for up to about 7 hr when studied by labelling with [ 14C]proline. The secretion of collagen [ 14C]hydroxyproline was also almost linear for up to 7 hr. Betamethasone-17-valerate decreased the total incorporation of [ 14C]proline and the decrease in the synthesis of [ 14C]hydroxyproline was larger than the decrease in the total incorporation, indicating that the collagen synthesis was affected more than other protein synthesis. Betamethasone-17-valerate had no effect on the activity of prolyl hydroxylase, when the cells were incubated with this steroid in vitro or, when the activity of the purified enzyme was assayed in the presence of this steroid. When the steroid was injected on to chorioallantoic membrane of embryonated eggs, the activity of prolyl hydroxylase decreased markedly in the tendon cells, indicating that in the system studied the amount of prolyl hydroxylase is decreased or that the enzyme is inactivated by this steroid in vivo.

Differential effects of hormones on precocious yolk sac retraction in chick embryos following administration by a new technique

Abstract A simple new technique allowing continuous application of hormones to the chorioallantoic membrane of the embryonated chicken egg is described. By use of this technique, by grafting the pars distalis, and by monitoring the hormonal response, with inhibitors and with induction of glucocorticoid-dependent liver enzymes, yolk sac retraction has been shown to be linked with thyroidal and not with adrenocortical or several other hormones. Evidence is also presented that the pars distalis, from chickens up to at least 18 months old, is capable of autonomous performance of certain adrenocorticotrophic or thyrotrophic functions when grafted on the chorioallantoic membrane of embryonated eggs.

The isolation and characterization of a new bovine herpesvirus associated with abortion

A virus isolated from the lung of an aborted Hereford fetus was shown to possess the physical and chemical properties of the herpesvirus group. The virus designated bovine herpesvirus (BH-1247) was isolated in cultures of Madin-Darby bovine kidney (MDBK) and primary bovine embryonic kidney (BEK) cells. Electron microscopic studies revealed typical forms of virus particles of the herpesvirus group. The virus was sensitive to chloroform and virus replication was inhibited by the addition of 5-iododeoxyuridine into the cell culture medium. The characteristic features of the cytopathic changes were syncytial formations with intranuclear inclusion bodies. Discrete plaques were formed in MDBK cell cultures overlaid with agar. Virus growth studies in BEK cells revealed infectious virus to be cell associated and replicated at low titer. By serum neutralization tests the virus was shown to be distinct from bovine herpesviruses; infectious bovine rhinotracheitis, DN-599, Movar 33/63, bovine mammallitis, malignant catarrhal fever and feline viral rhinotracheitis, equine herpesvirus I and pseudorabies. The isolate was nonpathogenic to mice inoculated subcutaneously, intracerebrally and intraperitoneally. Virus replication was not demonstrated when inoculated on the chorioallantoic membrane of embryonated eggs.

Characteristics of a virus causing a pox disease in sheep and goats in Kenya, with observations on the epidemiology and control

The sheep and goat pox viruses isolated in Kenya are not host specific as in the Middle East and India. The virus strains from one species are of a similar pathogenicity for the other, and the same virus appears to occur in the field outbreaks in mixed flocks. A silent infection with no skin lesions occurs in the field outbreaks. The virus grows well in lamb testis cell cultures, it does not haemadsorb nor haemagglutinate and is ether sensitive. Most strains could not readily be adapted to grow on the chorioallantoic membrane of embryonated eggs; one isolation only was made in this way. Staining of the virus inclusions showed that they were of DNA; the virus resembled that of lumpy skin disease in many characteristics. It was possible to cross-protect both species with virus of sheep or goat origin. A vaccine was made from tissue culture virus adsorped onto aluminium hydroxide gel and treated with formalin.

Structural and growth characteristics of two avian reoviruses

Two virus strains which had been suspected to be the etiological agents of infectious bursitis (Gumboro disease) and of inclusion body hepatitis of chickens were characterized by their morphology, their peptide composition and the segmented genome of their double-stranded RNA to be typical reoviruses. Although the 2 avian strains did not clearly differ in their serological behaviour, the size of some of their RNA segments were not identical. Both strains replicated in tissue cultures prepared from the chorioallantoic membrane of embryonated eggs with growth characteristics of reoviruses.

SUBTYPING OF HERPES SIMPLEX VIRUS

A number of different techniques to be used for the subtyping of herpes simplex virus (HSV) strains were studied. The strains were inoculated on chorio-allantoic membranes of embryonated eggs and on green monkey kidney (GMK) cells in order that the morphology of plaques produced might be observed. They were classified serologically by determination of K-values and inoculated intracerebrally in mice in order that their pathogenicity for mice might be observed. The inhibitory effect of high concentrations of thymidine on the multiplication of the strains in GMK cells cultures and the heat-stability of the virus-induced thymidine kinases were investigated. Rates of inactivation of the strains in the presence of AgNO3 were compared and, finally, the association of focal liver necrosis in intraperitoneally inoculated mice with the results of the serological typing was observed. The results suggested that the liver necrosis test was simple as well as accurate and useful as a screening typing-test. Reliable results were also obtained serologically and by the method demonstrating differences in the heat-stability of viral thymidine kinase. Using the other methods studied, difficulties to obtain clear-cut or reproducible typing results were encountered.

Preparation and Purification of Merozoites of Eimeria tenella

Second-generation merozoites of Eimeria tenella were obtained from both infected cecal tissue and infected chorioallantoic membranes of embryonated eggs. The merozoites were harvested from the tissue by incubation with hyaluronidase, yielding approximately 4 times 10(7) merozoites per cecum and 3 times 10(6) merozoites per chorioallantoic membrane. Subsequent purification of the merozoites by density centrifugation and glass bead filtration resulted in a 54% overall yield and a final preparation of approximately 95% purity. The viability of such preparations was established by inoculation of the merozoites to the ceca of chickens, resulting in oocyst production by 48 hr. This purification procedure allows for a rapid preparation of E. tenella during its second asexual stage in sufficient quantity and purity for biochemical study.

Recovery of a new herpesvirus from the ground squirrel (Citellus citellus).

A new herpesvirus recovered from the kidneys of mature ground squirrels (Citellus citellus) produced an effect in tissue culture cells resembling that of herpesviruses, including the formation of multinucleated syncytia and type A intranuclear inclusions. The isolate caused latent infection in the natural host, but it proved fatal for intracerebrally inoculated suckling mice and it produced pocks on the chorioallantoic membrane of embryonated eggs. Electron-microscopic examination of infected cells revealed intranuclear virus particles exhibiting a size and ultrastructure characteristic of herpesviruses. The isolate was ether-resistant and a DNA nucleic acid type was inferred from observations on inhibition by fluorinated pyrimidine. This ground squirrel herpesvirus was found to be antigenically distinct from Herpesvirus hominis and from ground squirrel cytomegalovirus, with no detectable cross-reactivity.

Preparation of Fowl Pox Vaccine on Chicken-Embryo-Dermis Cell Culture

SUMMARY A chicken-embryo-dermis (CED) cell-culture system derived from the skin of a specific-pathogen-free embryo is defined and shown to be highly susceptible to the fowl pox virus (FPV). The high susceptibility of the system and the growth characteristics of the virus suggest that the host-tissue specificity for FPV persists after the cells have differentiated in cell cultures. The cell system consists of cell strains derived from primary CED cell cultures. A vaccinal strain of FPV of Canadian origin (CFPV) was adapted to the cell system, and an experimental batch of vaccine was prepared from the 5th passage and tested. The cell-culture vaccine has higher biological properties than a conventional CFPV vaccine prepared on the chorioallantoic membrane of embryonated eggs. The advantages of the cell-culture FPV vaccine are discussed. The behavior and requirements of the CED cell system are described.

Intrauterine herpes simplex infection

A 25-year-old woman, para 2, gravida 3, was first examined by her physician on November 17, 1970, at approximately 20 weeks’ gestation. Previous pregnancies were uncomplicated. She denied ever having recurrent “cold sores.” Her husband had no history of penile lesions. In September, 1970, the patient was treated for a transient vaginal discharge by another physician. She had sustained minor bruises in an automobile accident in July, 1970. Complete physical examination was normal. The mouth, perineum, vagina, and cervix were free of lesions. Venereal Disease Research Laboratory test was nonreactive. Reexamination on January 21,.1971, was normal. On January 23, 1971, the patient was hospitalized with ruptured membranes of 12 hours’ duration. Spontaneous labor developed. A wellformed 4 pound male infant was delivered after a labor of 6% hours. Apgar score was 8 at one minute. Examination of the infant was normal except for the skin (Figs. I and 2). Numerous grouped and isolated clear tense vesicles on reddened bases were concentrated on the extremities and abdominal skin. Erosions, scales, and scars were present. There was a 2 by 3 cm. scar to the right of the umbilicus. The mouth and eyes were clear. Herpes simplex infection was immediately suspected. Consequently, a thorough examination of the cervix, vagina, and perineum was performed for herpetic lesions. None were found. Smears and skin biopsy revealed unilocular, intraepidermal vesiculation, containing acantholytic, multinucleated cells with a condensed peripheral cytoplasm. Feulgen-negative, eosinophilic inclusion bodies confined to the nuclei were visualized. Herpes simplex virus was isolated in the tissue culture from the vesicle fluid. Inoculation of the isolate onto the chorioallantoic membrane of embryonated eggs produced pocks characteristic of the Type 1 strain. The virus was sensitive to iododeoxyuridine in vitro. Serum bilirubin was 12 mg. per cent. Immunoglobulin A was 6, Immunoglobulin M was 25, and Immunoglobulin G was 640. Complete blood count, differential, electroyltes, bacterial blood and skin cultures, throat cultures, urinalysis, and chest roentgenogram were normal. Treatment consisted of 150 mg. of iododeoxyFig, I. Infant at birth showing grouped and isolated herpetic vesicles in various stages of development.

Characteristics of a conditional mutant of Rous sarcoma virus defective in ability to transform cells at high temperature

A conditional mutant of SR-RSV-D with a highly reduced ability to transform chicken cells at high temperature (41°) was obtained by mutagenization with 5-fluorouracil. This ts mutant (FU-19) retains its capacity to replicate at 41°C whereas it produces 30- to 50-fold fewer foci of Rous cells in monolayer cultures at this temperature than at 37°. Cells grown at 41° transform to Rous cells within a few hours after being shifted to 37°, and cells transformed at 37° revert also within a few hours to a normal morphology and behavior after being shifted to 41°. Hence, maintenance of the transformed state depends on the continuous expression of some specific viral gene(s). When high molecular weight dextran sulfate is added to the medium, the number of residual foci produced at the restrictive temperature, or still observed after shifting to this temperature, is considerably increased. This suggests that the expression of the transforming viral gene(s) alters the cell surface even at 41°—the mutant virus being leaky—and that this alteration produces transformation in a higher number of cells when cellular adhesion is reduced by polyanions. On the other hand, transformation after shifting to 37° requires only protein synthesis, i.e., presumably translation of the transforming information of viral RNA, whereas “detransformation” after shifting to 41° apparently requires no macromolecular synthesis. FU-19 also fails to produce tumors at 41° on the chorioallantoic membrane of embryonated eggs, and sarcoma cells produced in the CAM at 37° disappear after shifting to 41°. Hence, transformation of chicken cells in vivo as well as in vitro is a reversible phenomenon and similarly depends on the continuous expression of the viral transforming gene(s).

Herpes virus aotus: a latent herpesvirus from owl monkeys (Aotus trivirgatus) isolation and characterization.

SummaryAn uninoculated batch of owl monkey kidney cell cultures yielded a viral agent 23 days after the cell culture was initiated. This agent possessed the physical, chemical, cytopathic, histological, and ultrastructural properties of a herpesvirus. Random testing of owl monkey sera showed the presence of high titered neutralizing antibodies against this new agent. Herpesvirus simplex, Herpesvirus T., Herpesvirus B, Herpesvirus suis, Herpesvirus saimiri, infectious bovine rhinotraecheitis, and Herpesvirus saguinus, OMKI 372, and OMKI 68-69 antisera failed to neutralize the infectivity of this new agent. In cell cultures the virus grew best in cells of owl monkey origin. It also grew (poorly) in cebus monkey kidney cell cultures, some batches of squirrel monkey kidney cells, in Vero, BSC-1, human embryonic lung, whole human embryo and human embryo skin and muscle cells. It failed to form plaques under an agar overlay and does not form pocks on chorioallantoic membranes of embryonated eggs. Based on these...

Herpes-like virus of the guinea pig: propagation in brain tissue of guinea pigs and mice.

In a previous paper, Hsiung and Kaplow reported the isolation of a herpes-like virus (HLV) from a strain of guinea pigs susceptible to leukemia [1]. This agent was found to grow in various tissues from the guinea pig and in cultures of rabbit kidney cells, but it did not multiply in other host systems that include monkey and hamster cells and the chorioallantoic membrane of embryonated eggs. Brain cells were not examined in these studies.

Studies on the antiviral effect of rifampicin in volunteers.

RIFAMPICIN1 is a clinically useful, orally active antibiotic2, synthesized from rifamycin SV(ref. 3), a product of Streptomyces mediterraneans. This antibiotic is effective in the treatment of a number of bacterial infections, especially in tuberculosis2,4; it has also been shown to inhibit the replication of poxviruses in tissue cultures5–9. Further studies have indicated that the antiviral effect of rifampicin is selective in that it does not affect uptake into cells of some precursors, and also in that viruses other than poxviruses can replicate in the presence of the drug5,10. The mechanism by which the selective antiviral effect is mediated seems to involve an inhibition of late viral protein synthesis11, virion assembly12 and possibly also the de novo synthesized viral polymerase13. More recent experiments (to be published) have shown that rifampicin inhibits the formation of lesions resulting from infection with vaccinia virus on the chorioallantoic membrane of embryonated eggs, and on the skin of rabbits, when the drug is locally applied.

The detection and characterization of an induced RNA polymerase in the chorioallantoic membranes of embryonated eggs infected with influenza A 2 virus

An induced RNA polymerase was identified in the microsome fraction of the chorioallantoic membranes of embryonated eggs infected with an influenza A 2 virus. The activity of the polymerase, as measured by the incorporation of GMP- 3H into RNA from GTP- 3H, reached a maximum at 6–8 hours post inoculation and was about 10-fold greater than the corresponding incorporation of the microsome fraction of uninfected membranes. The polymerase was dependent upon the presence of Mg 2+ ions and all four ribonucleoside triphosphates for maximum activity and was not inhibited by DNase or actinomycin D. The RNA synthesized in vitro by the polymerase was shown to be a heteropolymer and to contain high molecular weight species sedimenting at 19 S and 14 S. These RNA species were similar in both sedimentation properties and nucleotide composition to the major RNA species extracted from the intact virus. The influenza A 2-induced RNA polymerase and its RNA product are discussed in relation to the function of the enzyme in the replication of influenza viruses.

Herpes simplex infection of the female genital tract. II. Association of cytopathic effect in cell culture with site of infection.

In our continuing effort to elucidate the relationship between infection of the female genital tract with herpes simplex virus (Herpesvirus hominis) and cervical carcinoma, we have been studying and comparing the biologic characteristics of strains of herpesvirus isolated from genital and nongenital sites. This relationship has also been investigated by Naib [1] and Rawls et al. [2]. Both groups found a positive correlation between evidence of genital infection with herpesvirus and the development of cervical neoplasia. A previous report [3] indicated that a group of approximately 500 women with a relatively high risk of developing cervical carcinoma had a 1.6% rate of genital infection with herpesvirus compared with an infection rate of less than 0.1% in approximately 1,900 normal women. It has been established that there are at least 2 antigenic types of human herpesvirus [4, 5]. It appears that the antigenic group of viruses referred to as type 1 is the cause of nongenital infections, whereas antigenic type 2 strains are responsible for herpes genitalis [6]. Genital strains can be differentiated from nongenital strains by their cytopathic effect in cultures of HeLa cells [7], rabbit kidney cells, and human lung fibroblast cells [8]. Genital and nongenital isolates also can be differentiated by their growth and thermal stability curves [8], by the size of pocks produced on the chorioallantoic membrane of embryonated eggs [9-11], and by their degree of neurovirulence in rabbits and mice [8]. This report describes a cytopathologic comparison of genital and nongenital strains of herpesvirus which were cultivated in human embryonic kidney cell cultures. Methods