Dramatic differences in the response of macrophages from B2 and B19 MHC-defined haplotypes to interferon gamma and polyinosinic:polycytidylic acid stimulation.

The chicken MHC has been associated with disease resistance, though the mechanisms are not understood. The functions of macrophages, critical to both innate and acquired immunity, were compared between the more infectious bronchitis virus-resistant B2 and the more infectious bronchitis virus-susceptible B19 lines. In vivo peripheral blood concentrations of monocytes were similar in B2 or B19 homozygous haplotypes. Peripheral blood-derived macrophages were stimulated with poly I:C, simulating an RNA virus, or IFNγ, a cytokine at the interface of innate and adaptive immunity. Not only did B2-derived peripheral monocytes differentiate into macrophages more readily than the B19 monocytes, but as determined by NO production, macrophages from B2 and B2 on B19 genetic background chicks were also significantly more responsive to either stimulant. In conclusion, the correlation with resistance to illness following viral infection may be directly linked to a more vigorous innate immune response.

A DNA vaccine expressing ENV and GAG offers partial protection against reticuloendotheliosis virus in the prairie chicken (Tympanicus cupido).

Recurring infection of reticuloendotheliosis virus (REV), an avian oncogenic gammaretrovirus, has been a major obstacle in attempts to breed and release the endangered Attwater's prairie chicken (Tympanicus cupido attwateri). The aim of this study was to develop a DNA vaccine that protects the birds against REV infection. A plasmid was constructed expressing fusion proteins of REV envelope (env) and VP22 of Gallid herpesvirus 2 or REV gag and VP22. Birds vaccinated with these recombinant plasmids developed neutralizing antibodies; showed delayed replication of virus; and had significantly less infection of lymphocytes, specifically CD4+ lymphocytes. Although the vaccine did not prevent infection, it offered partial protection. Birds in field conditions and breeding facilities could potentially benefit from increased immunity when vaccinated.

Host Gene Expression of Macrophages in Response to Feline Coronavirus Infection.

Feline coronavirus is a highly contagious virus potentially resulting in feline infectious peritonitis (FIP), while the pathogenesis of FIP remains not well understood, particularly in the events leading to the disease. A predominant theory is that the pathogenic FIPV arises from a mutation, so that it could replicate not only in enterocytes of the intestines but also in monocytes, subsequently systemically transporting the virus. The immune status and genetics of affected cats certainly play an important role in the pathogenesis. Considering the importance of genetics and host immune responses in viral infections, the goal of this study was to elucidate host gene expression in macrophages using RNA sequencing. Macrophages from healthy male cats infected with FIPV 79-1146 ex vivo displayed a differential host gene expression. Despite the virus uptake, aligned viral reads did not increase from 2 to 17 h. The overlap of host gene expression among macrophages from different cats was limited, even though viral transcripts were detected in the cells. Interestingly, some of the downregulated genes in all macrophages were involved in immune signaling, while some upregulated genes common for all cats were found to be inhibiting immune activation. Our results highlight individual host responses playing an important role, consistent with the fact that few cats develop feline infectious peritonitis despite a common presence of enteric FCoV.

Improved protein detection using cold microwave technology.

Protein screening/detection is an essential tool in many laboratories. Owing to the relatively large time investments that are required by standard protocols, the development of methods with higher throughput while maintaining an at least comparable signal-to-noise ratio would be highly beneficial to many researchers. This chapter describes how cold microwave technology can be used to enhance the rate of molecular interactions and provides protocols for dot blots, western blots, and ELISA procedures permitting a completion of all incubation steps (blocking and antibody steps) within 45 min.

Cross-sectional quantitative RT-PCR study of feline coronavirus viremia and replication in peripheral blood of healthy shelter cats in Southern California.

Objectives The objectives of this study were to determine the prevalence of feline coronavirus (FCoV) viremia, and its replication in peripheral blood using quantitative RT-PCR (qRT-PCR) methodology in a population of 205 healthy shelter cats in Southern California, as well as to assess any possible connection to longitudinal development of feline infectious peritonitis (FIP). Methods The study was performed on buffy-coat samples from EDTA-anticoagulated whole blood samples of 205 healthy shelter cats. From 50 of these cats, fecal samples were also examined. FCoV genomic and subgenomic RNA in the buffy coats was amplified by a total FCoV RNA qRT-PCR. Evidence for FCoV replication in peripheral blood and feces was obtained by M gene mRNA qRT-PCR. Results Nine of 205 cats (4.4%) were viremic by the total FCoV RNA qRT-PCR, and one of these cats had evidence of peripheral FCoV blood replication by an FCoV mRNA qRT-PCR. The single cat with peripheral blood replication had a unique partial M gene sequence distinct from positive controls and previously published FCoV sequences. Neither seven of the nine viremic cats with follow-up nor the single cat with replicating FCoV with positive qRT-PCR results developed signs compatible with FIP within 6 months of sample collection. Conclusions and relevance FCoV viremia and peripheral blood replication in healthy shelter cats have a low prevalence and do not correlate with later development of FIP in this study population, but larger case-control studies evaluating the prognostic accuracy of the qRT-PCR assays are needed.

Macrophages from disease resistant B2 haplotype chickens activate T lymphocytes more effectively than macrophages from disease susceptible B19 birds.

Resistance to respiratory pathogens, including coronavirus-induced infection and clinical illness in chickens has been correlated with the B (MHC) complex and differential ex vivo macrophage responses. In the current study, in vitro T lymphocyte activation measured by IFNγ release was significantly higher in B2 versus B19 haplotypes. AIV infection of macrophages was required to activate T lymphocytes and prior in vivo exposure of chickens to NP AIV plasmid enhanced responses to infected macrophages. This study suggests that the demonstrated T lymphocyte activation is in part due to antigen presentation by the macrophages as well as cytokine release by the infected macrophages, with B2 haplotypes showing stronger activation. These responses were present both in CD4 and CD8 T lymphocytes. In contrast, T lymphocytes stimulated by ConA showed greater IFNγ release of B19 haplotype cells, further indicating the greater responses in B2 haplotypes to infection is due to macrophages, but not T cells. In summary, resistance of B2 haplotype chickens appears to be directly linked to a more vigorous innate immune response and the role macrophages play in activating adaptive immunity.

RNA sequencing demonstrates large-scale temporal dysregulation of gene expression in stimulated macrophages derived from MHC-defined chicken haplotypes.

Discovering genetic biomarkers associated with disease resistance and enhanced immunity is critical to developing advanced strategies for controlling viral and bacterial infections in different species. Macrophages, important cells of innate immunity, are directly involved in cellular interactions with pathogens, the release of cytokines activating other immune cells and antigen presentation to cells of the adaptive immune response. IFNγ is a potent activator of macrophages and increased production has been associated with disease resistance in several species. This study characterizes the molecular basis for dramatically different nitric oxide production and immune function between the B2 and the B19 haplotype chicken macrophages.A large-scale RNA sequencing approach was employed to sequence the RNA of purified macrophages from each haplotype group (B2 vs. B19) during differentiation and after stimulation. Our results demonstrate that a large number of genes exhibit divergent expression between B2 and B19 haplotype cells both prior and after stimulation. These differences in gene expression appear to be regulated by complex epigenetic mechanisms that need further investigation.

Phylogenetic analyses indicate little variation among reticuloendotheliosis viruses infecting avian species, including the endangered Attwater's prairie chicken.

Reticuloendotheliosis virus infection, which typically causes systemic lymphomas and high mortality in the endangered Attwater's prairie chicken, has been described as a major obstacle in repopulation efforts of captive breeding facilities in Texas. Although antigenic relationships among reticuloendotheliosis virus (REV) strains have been previously determined, phylogenetic relationships have not been reported. The pol and env of REV proviral DNA from prairie chickens (PC-R92 and PC-2404), from poxvirus lesions in domestic chickens, the prototype poultry derived REV-A and chick syncytial virus (CSV), and duck derived spleen necrosis virus (SNV) were PCR amplified and sequenced. The 5032bp, that included the pol and most of env genes, of the PC-R92 and REV-A were 98% identical, and nucleotide sequence identities of smaller regions within the pol and env from REV strains examined ranged from 95 to 99% and 93 to 99%, respectively. The putative amino acid sequences were 97-99% identical in the polymerase and 90-98% in the envelope. Phylogenetic analyses of the nucleotide and amino acid sequences indicated the closest relationship among the recent fowl pox-associated chicken isolates, the prairie chicken isolates and the prototype CSV while only the SNV appeared to be distinctly divergent. While the origin of the naturally occurring viruses is not known, the avian poxvirus may be a critical component of transmission of these ubiquitous oncogenic viruses.

The use of flow cytometry to discriminate avian lymphocytes from contaminating thrombocytes.

Evaluation of peripheral blood mononuclear cells (PBMC) in avian species by flow cytometry is complicated by the presence of large numbers of nucleated thrombocytes. With light scattering properties similar to those of lymphocytes, variations in the proportion of thrombocytes in PBMC can result in apparent shifts in percentages of lymphocyte subpopulations. We have applied a dual-labeling procedure for flow cytometric analyses to exclude thrombocytes from the analyzed population by labeling with K55 monoclonal antibody (MAb), which differentially labeled leukocyte and thrombocyte populations. Flow cytometric analyses with K55 labeled chicken PBMC differentiated leukocytes into three populations according to their intensity of fluorescence. Using the Kl MAb, the K55-low population was shown to consist of thrombocytes. Dual-labeling with K55 MAb and MAb specific for B lymphocyte, CD4 or CD8 markers indicated that the K55 intermediate population consisted of lymphocytes. Therefore, concentrations of CD4+ and CD8+ T lymphocytes could be determined from the lymphocyte fraction by gating specifically on the K55 intermediate cells. Selecting cross-reactive chicken MAbs that included K55, this protocol was shown to identify CD4+ and CD8+ T lymphocytes in PBMC of another avian species, the endangered Attwater's prairie chicken.

Pathogenesis of a Texas feline immunodeficiency virus isolate: an emerging subtype of clade B.

We have recently provided evidence that Texas feline immunodeficiency virus (FIV-TX) isolates are an emerging subtype sharing a common ancestry with clade B isolates. Specific, pathogen-free cats were infected, intravenously, with 500, 2000 or 8000TCID(50) of the FIV-TX53 virus to study the acute stage of infection. Infection of cats resulted in lymphadenopathy at 10 days post-infection (p.i.). By 7 weeks p.i., gag specific antibody could be detected from sera of all infected cats. Virus could be detected by culturing PBMC and by nested capsid PCR. A reduction in the absolute numbers of lymphocytes and neutrophils was observed in infected cats although there was no trend identified between this reduction and the viral dose administered. By 11 weeks p.i., the CD4(+)/CD8(+) T cell ratios from all infected cats had dropped from approximately 2 to below 1. While decrease in the ratio was dependent on the viral dose, the T cell ratios of cats receiving the highest dose had significantly dropped below 1 by 4-7 weeks p.i. This decrease in the ratio was accompanied by a sharp and temporal decline in the absolute CD4(+) T cells and a slight increase in the absolute CD8(+) T cell numbers with a dramatic expansion of cells with CD8beta(low) chain expression.

Experimental infection of Attwater's/greater prairie chicken hybrids with the reticuloendotheliosis virus.

Reticuloendotheliosis virus (REV), a common pathogen of poultry, has been associated with runting and neoplasia in an endangered subspecies of grouse, the Attwater's prairie chicken. The pathogenesis of REV infection was examined in experimentally infected prairie chickens. Three groups of four Attwater's/greater prairie chicken hybrids were infected intravenously with varying doses (tissue culture infective dose [TCID50], 200, 1000, and 5000) of a prairie chicken-isolated REV. A fourth group of four birds was not infected. Blood was collected prior to infection, and at various times up to 37 wk following infection. Peripheral blood mononuclear cells were examined for integrated proviral DNA by a single-amplification polymerase chain reaction (PCR) and nested PCR of a region within the pol gene. The nested PCR identified REV proviral DNA in all REV-inoculated birds by 2 wk postinfection and confirmed chronic infection throughout the study. With the exception of a bird that died from bacterial pneumonia 8 wk postinfection, neoplasia, resembling that seen in naturally occurring infections, was observed in all birds, even those receiving as little as 200 TCID50 of virus.

Specific antibody secreting cells from chickens can be detected by three days and memory B cells by three weeks post-infection with the avian respiratory coronavirus.

Infectious bronchitis virus (IBV), the first coronavirus described, has been a continuing problem in poultry for more than 70 years. IBV, causing a highly contagious respiratory disease in chickens, resembles the recently described severe acute respiratory syndrome virus in pathogenesis and genome organization. While previous studies demonstrated that effector and memory CD8(+) T lymphocytes are critical in controlling acute IBV infection and disease in chickens, here chicken anti-IBV antibody (IgG) secreting cells (ASC) in both peripheral blood mononuclear cells (PBMC) and spleens collected following IBV Gray infection were evaluated using an ELISPOT assay. The ASC in peripheral blood and spleens can be detected from 3 to 7 days post-infection (p.i.), which is 3-7 days earlier than anti-IBV IgG detected in the serum. The ASC frequency reached a maximum at 7-10 days p.i., and decreased more than 90% in the spleen and 70% in PBMC by 14 days p.i. The ASC levels in the PBMC then decreased gradually to 0.5 ASC/10(6) over the next 8 weeks. The higher concentration of about 20 ASC/10(6) cells in spleens may, at least partially, account for the presence of antibody in the serum although bone marrow ASC were not determined. In vitro stimulation of PBMC and splenocytes with IBV antigen demonstrated that memory B cells can be activated to secrete antibody by 3 weeks p.i. ELISPOT detection of primary B cells could be useful in the early detection of infection following infection with respiratory coronaviruses.

Feline CD8+ cells induced with FIV infected, irradiated T cells produce multiple anti-FIV factors.

Feline immunodeficiency virus (FIV) infection in cats is the only non-primate, small animal model for HIV-AIDS. Replication of FIV has been shown to be optimally suppressed by soluble factors produced by inducer cell-stimulated feline CD8+ cells from FIV-infected cats. The nature of this dose-dependent suppression of FIV was examined. Antiviral factors, produced in serum-free medium, were shown to be either heat stable or heat labile. Suppressing activity was identified in a heparin-bound fraction and the non-bound fraction and in fractions separated by reverse-phase HPLC. The FIV suppression could not be correlated with IFN type I or II. Neither alpha nor beta chemokines were likely candidates because molecular size exclusion centrifugation indicated that the major factors were larger than 50 kD. Identified qualitative differences in the properties of the soluble suppressive activity generated from feline lymphocytes indicated that multiple factors are responsible for the non-cytolytic CD8+ T cell suppression of FIV replication.

Feline B7.1 and B7.2 proteins produced from swinepox virus vectors are natively processed and biologically active: potential for use as nonchemical adjuvants.

Costimulatory ligands, B7.1 and B7.2, have been incorporated into viral and DNA vectors as potential nonchemical adjuvants to enhance CTL and humoral immune responses against viral pathogens. In addition, soluble B7 proteins, minus their transmembrane and cytoplasmic domains, have been shown to block the down regulation of T-cell activation through blockade of B7/CTLA-4 interactions in mouse tumor models. Recently, we developed swinepox virus (SPV) vectors for delivery of feline leukemia antigens for vaccine use in cats [Winslow, B.J., Cochran, M.D., Holzenburg, A., Sun, J., Junker, D.E., Collisson, E.W., 2003. Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin. Virus Res. 98, 1-15]. To explore the use of feline B7.1 and B7.2 ligands as nonchemical adjuvants, SPV vectors containing full-length feline B7.1 and B7.2 ligands were constructed and analyzed. Full-length feline B7.1 and B7.2 produced from SPV vectors were natively processed and costimulated Jurkat cells to produce IL-2, in vitro. In addition, we explored the feasibility of utilizing SPV as a novel expression vector to produce soluble forms of feline B7.1 (sB7.1) and B7.2 (sB7.2) in tissue culture. The transmembrane and cytoplasmic regions of the B7.1 and B7.2 genes were replaced with a poly-histidine tag and purified via a two-step chromatography procedure. Receptor binding and costimulation activity was measured. Although feline sB7.1-his and sB7.2-his proteins bound to the human homolog receptors, CTLA-4 and CD28, both soluble ligands possessed greater affinity for CTLA-4, compared to CD28. However, both retained the ability to partially block CD28-mediated costimulation in vitro. Results from these studies establish the use of SPV as a mammalian expression vector and suggest that full-length-vectored and purified soluble feline B7 ligands may be valuable, nonchemical immune-modulators.

Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin.

The host range of swinepox virus (SPV) is restricted to swine, although SPV has been shown to infect mammalian, non-swine cells, without recovery of infectious virus. SPV is a reasonable candidate for development as a non-productively replicating viral vector for use in non-swine, mammalian species, such as the cat. A novel SPV gene deletion (SPV 043) was created and found to be non-attenuating. This deletion was utilized to generate a stable recombinant virus expressing the Gag-Pro and Env proteins of feline leukemia virus (FeLV). Expression and replication of this vector was studied in embryonic swine kidney cells (ESK-4), and two feline cell lines, Crandell feline kidney cells (CRFK) and feline skin fibroblasts (FSF). Our results showed that feline cells were susceptible to infection by SPV and supported expression of foreign genes driven by synthetic poxvirus promoters, however, SPV viral DNA was not replicated in feline cells and infectious virus was not recovered. In addition, FeLV Gag virus-like particles were produced from both ESK-4 and CRFK cells and foreign antigens were incorporated into infectious SPV intracellular mature virions (IMV). These results suggest that SPV may have potential as a safe vaccine delivery vector for cats.

Comparisons of envelope through 5B sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes.

A 1.78 kb sequence, including the E, M, 5a and 5b genes, and the intergenic region between the M and 5a genes, of six US strains of infectious bronchitis (corona)virus (IBV) were sequenced and compared to the published sequences for two additional strains. The overall identities as determined through pairwise analyses of nucleotide sequences of the entire 1.78kb region ranged from 90 to 99%, with the 5b open reading frame (ORF) having the greatest identity (94-99%) while the identities of the E, 5a and M ORFs ranged from 87 to 100%. Nucleotide sequencing of recent field isolates from Alabama (Ala1) and California (Cal3) revealed distinct shifts in homology in the M gene, indicating two apparent recombination events between the Holland 52/Mass41-like strain and an Ark-like strain, both origins of commonly used vaccine strains. Putative sites of recombination could also be identified in both the E and M ORFs of laboratory strains of IBV.

Evaluation of cytokine gene expression in porcine spleen cells, peripheral blood mononuclear cells, and alveolar macrophages by competitive RT-PCR.

Cytokines act as an important regulator of immune responses. Since cytokine expression levels are generally very low, more accurate and reliable methods of measuring their expression are needed. In this study, a modified competitive reverse transcription-polymerase chain reaction assay was developed to determine the expression levels and patterns of porcine IFN-gamma, IL-2, IL-4, IL-10, IL-12 p 35, and IL-12 p40 in spleen cells, peripheral blood mononuclear cells (PBMC), and alveolar macrophages that were stimulated for 4 h by lipopolysaccharide or phytohemagglutinin. Of these cytokines, the expression level of IFN-gamma was the highest in all examined cells. Constitutive expression of IL-2 and IL-4 was demonstrated in spleen cells and PBMC stimulated with phytohemagglutinin. However, their expression extent was not determinable or extremely low in the lipopolysaccharide-stimulated spleen cells and alveolar macrophages. Moderately high IL-10 expression was observed in all examined cells. IL-12 p 35 expression in alveolar macrophages was always higher than in spleen cells and PBMC. IL-12 p40 expression in alveolar macrophages was higher than in PBMC, but was lower than in spleen cells. In spleen cells, the expression of IL-12 p40 was higher than that of IL-12 p 35. In alveolar macrophages and PBMC, however, IL-12 p 35 showed a higher expression than IL-12 p40. These results indicate that each cytokine has its own characteristic expression profile in different immune cells.

B7+CTLA4+ T cells engage in T-T cell interactions that mediate apoptosis: a model for lentivirus-induced T cell depletion.

Apoptosis in lymph node (LN) T cells of feline immunodeficiency virus (FIV)-infected cats is associated with cells co-expressing B7.1 and B7.2 costimulatory molecules, and their ligand CTLA4. To study the possibility of B7.1/B7.2-CTLA4 mediated T-T interactions and the predicted induction of T cell apoptosis in vitro, costimulatory molecules were up-regulated on CD4+ and CD8+ T cells by mitogen stimulation. B7.1 expression on in vitro stimulated CD4+ and CD8+ cells increased within 24h; B7.2 and CTLA4 expression increased after 48-72 h. Apoptosis, as analyzed by terminal deoxynucleotidyl transferase (transferase nick end labeling, TUNEL)-based staining followed by three color flow cytometric analysis, correlated to the cells expressing B7 and/or CTLA4. Blocking experiments revealed that CD4+ and CD8+ T cell apoptosis could be significantly inhibited with anti-B7 antibodies. As FIV infection results in immune activation with a T cell phenotype similar to that of the in vitro activated T cells, the data support the hypothesis that the chronic expansion of B7+CTLA4+ LN T cells in infected cats allows for T-T cell interactions resulting in T cell depletion and eventually the development of AIDS.

Spontaneous T cell apoptosis in feline immunodeficiency virus (FIV)-infected cats is inhibited by IL2 and anti-B7.1 antibodies.

Lymph node (LN) T cells from feline immunodeficiency virus (FIV)-infected cats have an increased expression of B7 co-stimulatory molecules as well as their ligand CTLA4, resembling an activation phenotype shown to induce anergy and apoptosis in activated T cells. In addition, LN T cells from FIV-infected cats also show increased spontaneous apoptosis compared to uninfected animals. The apoptosis observed in these animals occurs primarily in T cells expressing B7 and CTLA4, suggesting a role for B7 and CTLA4 interactions in the induction of anergy/apoptosis. In order to investigate the role of B7 and CTLA4 interactions on T cell apoptosis in LN T cells from FIV-infected cats, we performed blocking experiments by measuring T cell apoptosis in LN T cell cultures treated with anti-feline B7.1, B7.2, and CTLA4 specific antibodies, as well as interleukin (IL)-2. The addition of IL2, the primary cytokine produced by B7/CD28 interactions, resulted in a significant decrease of T cell apoptosis in cultured LN cells as assessed by two-color flow cytometry and TUNEL assay. The addition of anti-B7.1 antibodies significantly inhibited T cell apoptosis in FIV-infected cats with low-level plasma viremia, while addition of anti-B7.2 and anti-CTLA4 antibodies had no affect. These results suggest a role of B7 signaling in the increased spontaneous apoptosis observed in LN T cells from FIV-infected animals.

Association of the chicken MHC B haplotypes with resistance to avian coronavirus.

Clinical respiratory illness was compared in five homozygous chicken lines, originating from homozygous B2, B8, B12 and B19, and heterozygous B2/B12 birds after infection with either of two strains of the infectious bronchitis virus (IBV). All chickens used in these studies originated from White Leghorn and Ancona linages. IBV Gray strain infection of MHC homozygous B12 and B19 haplotype chicks resulted in severe respiratory disease compared to chicks with B2/B2 and B5/B5 haplotypes. Demonstrating a dominant B2 phenotype, B2/B12 birds were also more resistant to IBV. Respiratory clinical illness in B8/B8 chicks was severe early after infection, while illness resolved similar to the B5 and B2 homozygous birds. Following M41 strain infection, birds with B2/B2 and B8/B8 haplotypes were again more resistant to clinical illness than B19/B19 birds. Real time RT-PCR indicated that infection was cleared more efficiently in trachea, lungs and kidneys of B2/B2 and B8/B8 birds compared with B19/B19 birds. Furthermore, M41 infected B2/B2 and B8/B8 chicks performed better in terms of body weight gain than B19/B19 chicks. These studies suggest that genetics of B defined haplotypes might be exploited to produce chicks resistant to respiratory pathogens or with more effective immune responses.