Replication of avian sarcoma viruses in chicken macrophages

Avian sarcoma viruses of subgroups B and C were able to replicate in chicken embryonic macrophages (derived from yolk sac) and in adult macrophages (obtained from peripheral blood), while avian sarcoma viruses of subgroups A and D were not. Infectious center assays indicated that the proportion of macrophages infected by viruses representative of subgroups B and C which registered as infectious centers was significantly lower than that of fibroblasts infected by the same viruses. Moreover, the comparative growth of avian sarcoma virus B77 (subgroup C) in fibroblasts and in yolk sac cells showed that macrophages have a reduced ability to replicate this virus. In addition, viruses of subgroups B and C and mainly helper-independent viruses of these two subgroups induced morphological alterations (formation of giant cells) and biochemical changes (increased rate of sugar transport), indicating that the infection of chicken macrophages by avian sarcoma viruses of subgroups B and C led to the malignant transformation of these cells.     

Activating effects of interferons, lymphokines and viruses on cultured chicken macrophages

Cultured chicken bone-marrow-derived and blood monocyte-derived macrophages could be activated by various treatments: (1) With crude lymphokine preparations obtained from Concanavalin A (ConA)-stimulated chicken spleen or thymus cell cultures: (2) with virus-induced interferons (IFN) from cultured chicken embryo fibroblasts or macrophages; (3) with inactivated reovirus or live infectious bursal disease virus (IBDV). Macrophage activity was expressed by cytostatic effects against lymphoblastoid target cells, and by morphological changes, such as enhanced spreading of the cells. The macrophage-activating capacity of lymphokine preparations (50% cytostasis-inducing dose) was closely associated with their antiviral activity (IFN units). According to its physico-chemical properties, ConA-induced lymphocyte interferon was considered to be IFN-gamma, but acid-and heat-resistant IFN-alpha or IFN-beta may also have been present in spleen cell or thymocyte culture supernatants. Virus-induced interferons (IFN-alpha/beta) were less effective in macrophage activation than in antiviral activity. Experimental results strongly suggested that macrophage activation by viruses was mediated by endogenous IFN.     

Susceptibility and resistance of chicken macrophages to avian RNA tumor viruses.

A set of trans dominant mutations in the cI repressor gene of λ bacteriophage was mapped with the aid of a set of deletions. The clustering of all the mutants in a restricted region of the repressor gene suggests that these mutants may alter a site of the repressor responsible for the recognition of the operator.     

Persistence of avian oncoviruses in chicken macrophages.

Inoculation of avian oncoviruses into 1- to 2-month old chickens led to a rapid production of antiviral humoral antibodies. Under these conditions it was found that avian leukosis viruses are sequestered in macrophages of peripheral blood, in which they can persist for a long period of time (up to about 3 years). In contrast, avian sarcoma viruses were never found in macrophages from chickens during the progression of sarcomas or after regression of the tumors.     

Growth inhibition of Marek's disease T-lymphoblastoid cell lines by chicken bone-marrow-derived macrophages activated in vitro

Studies have been performed on the induction of cytostatic activity of cultured chicken bone-marrow-derived macrophages. Cultured macrophages were exposed to supernatants of ConA-stimulated spleen cell cultures (lymphokines), lipopolysaccharide (LPS), ConA or infectious bursal disease virus (IBDV). Cytostatic activity of macrophages was examined by testing their growth-inhibiting effect on T-lymphoblastoid Marek's disease cell lines RP-1, HP-2 and MSB-1 as target cells. Lymphokines, LPS and IBDV caused considerable enlargement of adherent macrophages. Results of cytostasis assays suggested that morphological signs of macrophage activation were not correlated with the cytostatic potency of activated macrophages. Lymphokine-activated macrophages caused at least an 80% growth inhibition of target cells, whereas LPS, ConA or IBDV-activated macrophages exhibited only a marginal cytostatic effect in the range of 20%. Attempts to detect soluble cytostatic factors in supernatants of activated macrophage cultures failed or had equivocal results. Untreated macrophages were rarely cytostatic at an effector:target cell ratio of I:1 to 4:1, and they stimulated RP-1 cell growth if these cells were seeded at suboptimal concentrations. The results suggest that the suppressor activity of macrophages from Marek's disease virus-infected chickens, as demonstrated by other authors in vitro, is probably a result of nonspecific immunomodulation in vivo where lymphokines may also play a major role.     

Analysis of immunoglobulins in chicken antibody to avian leucosis viruses

Fractionations of chicken sera containing antibody to the avian leucosis viruses, RAV—1 or RAV—6 were carried out. A small proportion of antibody activity was found in the serum IgM fractions from the majority of birds, but most of the antibody activity was recovered in the serum IgG fractions.     

Effects of ovotransferrin on chicken macrophages and heterophil-granulocytes

Ovotransferrin (OTF) is an acute phase protein in chickens, serum levels of which increase in inflammation and infections. To understand the significance of OTF in inflammation, we studied its in vitro effects on HD11 cells, a macrophage cell line, and heterophils isolated from blood using a panel of variables indicative of cellular activation. These included the production of interleukin-6 (IL-6), nitrite, matrix metalloproteinase (MMP), oxidation of dichlorofluorescein diacetate for respiratory burst and the degranulation of heterophils by the loss of fluorescein isothiocyanate positive cytoplasmic granules. The results show that ovotransferrin stimulates the production of IL-6, nitrite and MMP by HD11 cells and augments phorbol ester-induced respiratory burst. Ovotransferrin stimulated heterophils to produce IL-6, and MMP, but failed to produce nitrite, enhanced respiratory burst activity and degranulation. These results suggest that ovotransferrin can modulate macrophage and heterophil functions in chickens.     

Replication of five bovine respiratory viruses in cultured bovine alveolar macrophages

The replication of five bovine respiratory viruses in cultured bovine alveolar macrophages (BAM) was investigated. The bovine herpesvirus DN-599 strain did not cause cytophatic effect (CPE), extracellular virus and intracellular antigen were not demonstrated. Although a small number of bovine respiratory syncytial virions were consistently released and about 1 percent of the BAM were fluorescence antibody (FA) positive the virus caused no CPE. Infectious bovine rhinotracheitis virus caused CPE at an input multiplicity of about 0.5, and 5 percent of the BAM were FA positive. Both bovine parainfluenza-3 and bovine viral diarrhea viruses caused CPE, infective virions were released and considerable proportions of the BAM were FA positive.     

Expression of Ia antigen in colonies of bone-marrow-derived macrophages

Cells in colonies of culture-derived mouse and rat bone marrow macrophages were examined for membrane Ia antigen to determine if the steady-state expression of the antigen was restricted to macrophages derived from a distinct population of progenitor cells. Multiple subcultures of macrophages derived from single soft-agar colonies were tested for Ia+ cells on three different days of culture. About one-half of the colonies gave rise to subcultures that never contained Ia+ cells, about 40% yielded subcultures that contained some Ia+ cells on at least 2 of the 3 assay days, and about 10% of the colonies produced subcultures that contained Ia-bearing cells on all 3 assay days. Thus, when cultures were assayed at any one time for their content of Ia-bearing cells, the results raised the possibility of phenotypically distinct subpopulations of progenitors. However, sequential analyses of the subcultures revealed the variable expression of Ia on cells from at least one-half of the colonies. We conclude that, under steady-state conditions, the presence of Ia antigen on bone marrow-derived macrophages is not clonally restricted. That a T-cell-derived lymphokine induced Ia antigen on essentially all the cells in most of the colonies of macrophages confirms their potential to express the antigen.     

Genetic susceptibility of chicken times quail hybrid embryos to avian RNA tumour viruses.

An attempt was made to hybridize the chicken (Gallus domesticus) male with Japanese quail (Coturnix coturnix japonica) female in order to study the genetic susceptibility of hybrid embryos to avian RNA tumour viruses of subgroups, A, B, D and E. In the hybrids the results supported the prevailing concept that susceptibility is dominant over resistance regardless of the dominant trait contributed by either parent. It was also observed that the Ie gene of the chicken was unable to suppress the 'quail-coded' susceptibility to subgroup E virus in the hybrid system, suggesting the lack of penetrance of the Ie gene. Despite the fact that some hybrids were resistant to viruses of subgroups B and D, they were susceptible to subgroup E virus, which was not expected on the basis of the concept that subgroup B-resistant cells cannot be E-susceptible. Also, the hybrids were susceptible to E virus regardless of gs antigen expression and presence of the Ie gene in the genome. This indicates that our earlier suggestion that the Ie gene is another expression of the gs antigen-determining gene is inconsistent.     

Effects of chicken embryo age on time to death following infection by avian influenza viruses: implications for distinguishing highly pathogenic isolates

When white leghorn (WL) chick embryos ranging in age from 8 to 13 days were inoculated with a variety of avian influenza virus (AIV) isolates, strain-specific differences in embryo mean death times (MDT) were observed. Non-highly pathogenic (nHP) strains killed 8 or 9 day-old embryos much more rapidly than 12 or 13 day-old embryos. Highly pathogenic (HP) strains, however, were less sensitive to embryo age resulting in similar MDTs in both older and younger embryos. These observations were consistent over a broad range of virus doses for both HP and nHP strains. When a HP derivative of H5N2 AIV was compared to its nHP parent, the derivative killed older embryos more rapidly than the parent virus, while MDTs in younger embryos were the same for both parent and derivative. The two strains further exhibited clear differences in the structure of their respective hemagglutinin, a previously described pathogenicity determinant for this virus. Thus it may be possible to readily demonstrate the HP phenotype in AIV strains based on MDT measurements in WL embryos.     

Growth of avian and human influenza viruses in organ cultures of duck and chicken colons

Summary Colons from ducks and chicken 1, 7, 14 and 28 days old maintained near-normal morphology up to 48 and 96 hours respectively in a system using NCTC 135 medium (1 part)+Dulbecco's modified Eagle's medium (9 parts), at 37° C and 95 per cent 0₂/5 per cent CO₂. In the colon of 1 and 28 day-old ducks, duck influenza virus (Hav7N2) and budgerigar influenza virus (Hav4Nav1) grew to peak titer by hour 72, whereas human influenza virus (H3N2) did not grow. In the colon of 1 day-old chicken, the three viruses grew in the order of first duck virus, then budgerigar virus and then human virus, but in the colon of 28 day-old chicken, the growth of human virus was much less. Specific fluorescence was demonstrated in the mucosal epithelium of the colon of ducks and chicken, and intensity of fluorescence correlated with virus yield. The fact that the avian and not the human influenza viruses showed good growth in the duck clons coincided with the fact that influenza viruses possessing avian hemagglutinin subtypes have frequently been isolated in nature from duck intestines.With 3 Figures     

TRIM21 controls Toll-like receptor 2 responses in bone-marrow-derived macrophages

TRIM21 is an interferon-stimulated E3 ligase that controls the activity of pattern-recognition signaling via ubiquitination of interferon regulatory factors and DDX41. Previous studies on the role of TRIM21 in innate immune responses have yielded contradictory results, suggesting that the role of TRIM21 is cell specific. Here, we report that bone-marrow-derived macrophages (BMDMs) generated from Trim21^−/− mice have reduced expression of mature macrophage markers. Reflecting their reduced differentiation in response to macrophage colony-stimulating factor (M-CSF), Trim21^−/− BMDMs had decreased expression of M-CSF signature genes. Although Trim21^−/− BMDMs responded normally to Toll-like receptor 9 (TLR9) activation, they produced lower levels of pro-inflammatory cytokines in response to the TLR2 agonist PAM3CSK4. In line with this, the response to infection with the Bacillus Calmette–Guérin strain of Mycobacterium bovis was also diminished in Trim21^−/− BMDMs. Our results indicate that TRIM21 controls responses to TLR2 agonists.     

Cleavability of hemagglutinin determines spread of avian influenza viruses in the chorioallantoic membrane of chicken embryo

Summary The spread of infection in the chorioallantoic membrane (CAM) has been analysed with pathogenic and non-pathogenic avian influenza A viruses. After allantoic inoculation of pathogenic strains, high titers of infectious virus were found in the allantoic fluid, and virus growth could be demonstrated by immunohistology and electron microscopy in the allantoic epithelium, the mesenchyma, and in the chorionic epithelium. By the same route of inoculation, non-pathogenic strains yielded also high titers of infectious virus in the allantoic fluid, but virus replication was restricted to the allantoic epithelium and did not occur in the other cell layers. After chorionic inoculation of pathogenic strains, replication occurred in all layers of the CAM, and infectious virus was released into the allantoic fluid. However, when the chorionic epithelium was infected with a non-pathogenic strain, infection did not spread beyond the site of inoculation. These differences in virus spread are based on differential activation of the hemagglutinin by proteolytic cleavage. The hemagglutinin of pathogenic strains is cleaved in cells of each layer, whereas the hemagglutinin of non-pathogenic strains is cleaved only in the allantoic epithelium. In epithelial cells, virus budding occurred nearly exclusively at the apical side of the cell surface, but this polarization of virus maturation was found with both pathogenic and nonpathogenic strains, indicating that it does not account for the differences in virus spread and, thus, in pathogenicity.With 4 Figures     

Role of avian pathogenic Escherichia coli virulence factors in bacterial interaction with chicken heterophils and macrophages

Avian pathogenic Escherichia coli (APEC) cause extraintestinal disease in avian species via respiratory tract infection. Virulence factors associated with APEC include type 1 and P fimbriae, curli, aerobactin, lipopolysaccharide (LPS), K1 capsular antigen, temperature-sensitive hemagglutinin (Tsh), and an uncharacterized pathogen-specific chromosomal region (the 0-min region). The role of these virulence factors in bacterial interaction with phagocytes was investigated by using mutants of three APEC strains, each belonging to one of the most predominant serogroups O1, O2, and O78. Bacterial cell interaction with avian phagocytes was tested with primary cultures of chicken heterophils and macrophages. The presence of type 1 fimbriae and, in contrast, the absence of P fimbriae, K1 capsule, O78 antigen, and the 0-min region promoted bacterial association with chicken heterophils and macrophages. The presence of type 1 and P fimbriae, O78 antigen, and the 0-min region seemed to protect bacteria against the bactericidal effect of phagocytes, especially heterophils. The tested virulence factors seemed to have a limited role in intracellular survival for up to 48 h in macrophages. Generally, opsonized and nonopsonized bacteria were eliminated to the same extent, but in some cases, unopsonized bacteria were eliminated to a greater extent than opsonized bacteria. These results confirm the important role of type 1 fimbriae in promotion of initial phagocytosis, but nevertheless indicate a role for type 1 fimbriae in the protection of bacteria from subsequent killing, at least in heterophils. The results also indicate a role for K1 capsule, O78 antigen, P fimbriae, and the 0-min region in initial avoidance of phagocytosis, but demonstrate an additional role for O78 antigen, P fimbriae, and the 0-min region in subsequent protection against the bactericidal effects of phagocytes after bacterial association has occurred.