Chicken interferon type I inhibits infectious bronchitis virus replication and associated respiratory illness.

Infectious bronchitis virus (IBV) causes an economically important respiratory disease in poultry worldwide. Previous studies have shown that CD8(+) cytotoxic T lymphocytes (CTL) are critical in controlling acute IBV infection, but the role of innate immunity is unknown. This study describes the in vitro and in vivo anti-IBV activity of natural spleen cell-derived and recombinant chicken interferon type I (rChIFN-alpha). Both natural and rChIFN-alpha inhibited replication of the Beaudette strain of IBV in chicken kidney cells (CKC) in a dose-dependent manner, with the antiviral activity of the former accounted for entirely by its content of type I IFN. IFN at 100 U/ml reduced viral replication by 50% as measured by syncytia formation. In addition, the spleen cell-derived supernatants (natural IFN) inhibited tracheal ring ciliostasis mediated by the Gray strain of IBV. Optimal protection against IBV-induced respiratory disease was obtained after intravenous or oral administration of ChIFN given 1 day before virus challenge and each of 5 days thereafter. ChIFN-I protected chicks from clinical illness by delaying the onset of the disease and decreasing the severity of illness, demonstrating its potential as an immune enhancer.

Dose response studies of acute feline immunodeficiency virus PPR strain infection in cats.

The effects of virus dose on host response were evaluated for the PPR strain of feline immunodeficiency virus (FIV-PPR). Specific pathogen-free cats were inoculated intravenously with 50, 250 or 1250 TCID(50) of FIV-PPR. Two weeks after inoculation, virus was detected in 10(6) peripheral blood mononuclear cells (PBMCs) of all infected animals, and the CD4(+):CD8(+) T lymphocyte ratios fell from greater than 2 to approximately 1 in all infected animals within the first 8 weeks after infection. Provirus detected in all groups using PCR and 10(3) PBMC was biphasic. Nine of 15 animals were positive between weeks 2 and 4 p.i. and 14 of 15 were positive by week 8 p.i. Transient lymphadenopathy was detected in most cats receiving 1250 TCID(50) and the 250 TCID(50) of virus, whereas no lymphadenopathy was detected in the 50 TCID(50) group or the five uninfected cats. Animals that had received the largest dose seroconverted earliest (on average at week 4.0) and those receiving the least seroconverted last (on average at week 5.6). Neither neutropenia nor lymphopenia were detected. FIV-specific CTL responses of memory effector cells could be detected in animals receiving all three doses but was highly variable among individual animals. Neurological manifestations determined after 15 weeks p.i. were observed in most infected cats, including two of the three that had received 50 TCID(50) of virus. However, the observed neurologic abnormalities were markedly less severe in the animals receiving the least amount of virus. Therefore, lymphadenopathy and neurologic signs of illness were less severe and seroconversion was slower in the animals that received the lowest dose compared with those receiving the 250 and 1250 TCID(50) doses of the FIV-PPR strain.

Molecular cloning and expression of feline CD28 and CTLA-4 cDNA.

Feline CD28 and CTLA-4 (CD152) cDNA were cloned from Con-A stimulated feline peripheral blood mononuclear cells (PBMC) by rapid amplification of cDNA end-PCR (RACE-PCR). Both CD28 and CTLA-4 proteins belong to the immunoglobulin superfamily (Ig SF) and are composed of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain. The open reading frame (ORF) of CD28 cDNA encoded a predicted protein of 221 amino acids and that of CTLA-4 cDNA encoded a predicted protein of 223 amino acids. The B7 ligands binding motif MYPPPY hexamer was found on the extracellular Ig V-like domains of both receptors and phosphatidylinositol 3-kinase (PI 3-kinase) binding motifs pYMNM for CD28 and pYVKM for CTLA-4 were identified in the cytoplasmic domains. Comparisons of amino acid sequences of feline proteins with known sequences of other species indicated that rabbit CD28 and CTLA-4 were most closely related and mouse molecules were the least conserved with feline molecules. Comparison of each domain of both molecules with that of other animals showed that the cytoplasmic domain of CTLA-4 was 100% conserved and that of CD28 was the most conserved domain. The cloned CD28 and CTLA-4 cDNA could be expressed in transfected mammalian cells. Expression of feline CD28 and CTLA-4 mRNA in freshly isolated feline PBMC was demonstrated by RT-PCR. Stimulation of PBMC with Con-A similarly increased the expression of both CD28 and CTLA-4 mRNA.

The amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3' genomic RNA.

Previous studies indicated that the nucleocapsid (N) protein of infectious bronchitis virus (IBV) interacted with specific sequences in the 3' non-coding region of IBV RNA. In order to identify domains in the N protein that bind to RNA, the whole protein (409 amino acids) and six overlapping fragments were expressed as fusion polypeptides with six histidine-tags. Using gel shift assays, the intact N protein and amino polypeptides, from residues 1 to 171 and residues 1 to 274, and carboxyl polypeptides, extending from residues 203 to 409 and residues 268 to 407, were found to interact with positive-stranded IBV RNA representing the 3' end of the genome. The two 32P-labeled probes that interacted with N and the amino and carboxyl fragments of N were RNA consisting of the IBV N gene and adjacent 3' non-coding terminus, and RNA consisting of the 155-nucleotide sequences at the 3' end of the 504-nt 3' untranslated region. In contrast, the polypeptide fragment from the middle region, residues 101-283, did not interact with these 3' IBV RNAs. The binding site in the amino region of N was either not present or only partially present in the first 91 residues because no interaction with RNA was observed with the polypeptide incorporating these residues. Cache Valley virus N expressed with a histidine tag, bovine serum albumin, and the basic lysozyme protein did not shift the IBV RNA. The lower molarities of the carboxyl fragment compared with residue 1-274 fragment needed for equivalent shifts suggested that the binding avidity for RNA at the carboxyl domain was greater than the amino domain.

Adoptive transfer of infectious bronchitis virus primed alphabeta T cells bearing CD8 antigen protects chicks from acute infection.

Infectious bronchitis virus (IBV) infection and associated illness may be dramatically modified by passive transfer of immune T lymphocytes. Lymphocytes collected 10 days postinfection were transferred to naive chicks before challenge with virus. As determined by respiratory illness and viral load, transfer of syngeneic immune T lymphocytes protected chicks from challenge infection, whereas no protection was observed in the chicks receiving the MHC compatible lymphocytes from uninfected chicks. Protection following administration of T lymphocytes could be observed in chicks with three distinct MHC haplotypes: B(8)/B(8), B(12)/B(12), and B(19)/B(19). Nearly complete elimination of viral infection and illness was observed in chicks receiving cells enriched in alphabeta lymphocytes. In contrast, removal of gammadelta T lymphocytes had only a small effect on their potential to protect chicks. The adoptive transfer of enriched CD8(+) or CD4(+) T lymphocytes indicated that protection was also a function primarily of CD8-bearing cells. These results indicated that alphabeta T lymphocytes bearing CD8(+) antigens are critical in protecting chicks from IBV infection.

Cytotoxic T lymphocytes are critical in the control of infectious bronchitis virus in poultry.

Various strains of infectious bronchitis virus (IBV) cause respiratory, kidney, enteric and reproductive illnesses in chickens, especially in newly hatched chicks. Assays have been developed to identify Gray strain IBV-specific cytotoxic T lymphocyte (CTL) responses using viral infected antigen presenting cells (APC) and using the Semliki Forest virus vector infected APC expressing individual viral polypeptides. It was shown that major histocompatibility complex restricted CTL are responsible for early control of IBV infection. The kinetics of viral load observed in the lungs and kidneys correlated with the level of IBV-specific CTL activity of effector cells prepared from spleens of infected chicks. Adoptive transfer of immune T cells to chicks prior to infection demonstrated that IBV primed CD8(+), alphabeta T lymphocytes could protect chicks from acute infection. CTL determinants in the viral particle can be mapped to the spike and nucleocapsid proteins but not to the membrane protein. The carboxyl terminus of the nucleocapsid protein houses an epitope(s) responsible for induction of CTL responses to IBV N protein. Inoculation of DNA plasmid expressing the carboxyl terminus of Gray strain N resulted in induction of CTL that cross-react with two distinct IBV strains. In addition, this potential DNA vaccine resulted in protection of chicks against acute infection.

Sequence analyses of feline B7 costimulatory molecules.

Using RT-PCR amplifications with mRNA from mitogen-stimulated feline peripheral blood mononuclear cells, cDNA of feline B7-1 (CD80) and B7-2 (CD86) were cloned. The cDNA were sequenced and putative translated protein sequences compared with known counterpart sequences. Hydrophilicity patterns of the feline CD80 and CD86 which were only 26.8% identical at the amino acid sequence were very distinct from each other, but similar to the putative human CD80 and CD86 proteins, respectively. The feline CD80 gene encoded a protein of 292 amino acids and the CD86 gene encoded a protein of 329 amino acids. Amino-terminal signal sequences, extracellular Ig V- and Ig C-like domains, transmembrane domains, and carboxyl cytoplasmic domains were identified in both molecules. Although the most conserved domain among the CD80 sequences was the Ig C-like domain, the most conserved domain among the CD86 sequences was the Ig V-like domain. Among the known sequences, the bovine CD80 and the porcine CD86 sequences available for comparisons were identified as most closely related to the feline CD80 (63.3%) and CD86 (67.5%), respectively. The mouse molecules were the least identical (43.6 and 43.6%, respectively) with the feline CD80 and CD86 proteins. The human CD80 and CD86 molecules were 56.3 and 57.0% identical with the feline molecules.

Anti-feline immunodeficiency virus (FIV) soluble factor(s) produced from antigen-stimulated feline CD8(+) T lymphocytes suppresses FIV replication.

Feline immunodeficiency virus (FIV) causes AIDS-like symptoms in infected cats. Concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC) from chronically FIV strain PPR-infected cats readily expressed FIV. In contrast, when PBMC from these animals were stimulated with irradiated, autologous antigen-presenting cells (APC), at least a 10-fold drop in viral production was observed. In addition to FIV-specific cytotoxic T lymphocytes, anti-FIV activity was demonstrated in the cell-free supernatants of effector T lymphocytes stimulated with APC. The FIV-suppressive activity was induced from APC-stimulated PBMC of either FIV-infected or uninfected cats but not from ConA-stimulated PBMC. Suppression of FIV strain PPR replication was observed for both autologous and heterologous feline PBMC, was dose dependent, and demonstrated cross-reactivity and cell specificity. It was also demonstrated that the anti-FIV activity originated from CD8(+) T lymphocytes and was mediated by a noncytolytic mechanism.

Evaluation of expression patterns of feline CD28 and CTLA-4 in feline immunodeficiency virus (FIV)-infected and FIV antigen-induced PBMC.

It is known that CD28, a positive costimulatory receptor, plays a very important role in inducing the optimal stimulation of T lymphocytes. CTLA-4 (CD152), however, acts as a negative regulator in T lymphocyte activation. The effect of an feline immunodeficiency virus (FIV) infection on the expression of feline CD28 and CTLA-4 was studied with FIV-infected and uninfected peripheral blood mononuclear cells (PBMC) using a competitive PCR assay. The nature of CD28 and CTLA-4 expression was also examined with fresh and antigen-stimulated PBMC. FIV infection induced a lower expression of CD28, but a higher expression of CTLA-4 in the infected PBMC than in the uninfected PBMC. Relatively high levels of CD28 expression were demonstrated in both the fresh and the antigen-stimulated PBMC. The expression level of CTLA-4 in the freshly isolated PBMC was rather low, however, FIV antigen stimulation induced a relatively high expression of CTLA-4 in feline PBMC.

Cytotoxic T lymphocyte responses to infectious bronchitis virus infection.

Cytotoxic T lymphocyte (CTL) activity to infectious bronchitis virus (IBV) was examined at regular intervals between 3 and 30 days post infection (p.i.). The maximal CTL lysis of target cells infected with IBV with 82% was detected at 10 days p.i. The specific CTL activity began to decrease only after viral loads, which peaked at day 8 p.i. in both kidneys and lungs, started to decline. Therefore, the CTL response correlated with elimination of acute infection. IgM antibody did not appear until day 10 and levels peaked at day 12 p.i. whereas IgG antibody titers were detectable only by day 15 p.i., but continued to increase exponentially until day 30 p.i., the last day examined. IBV specific CTL epitope(s) were mapped within the carboxyl terminal 120 amino acids of nucleocapsid protein. In vivo inoculation of this fragment, as cDNA, induced protection against acute infection. The absence of viral neutralizing epitopes on the nucleocapsid protein would suggest that protection with known CTL eptiope(s) can be induced in the absence of neutralizing antibody.

Recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus.

The nucleocapsid protein of the Gray strain of infectious bronchitis virus (IBV) is highly immunogenic and cross-reactive among various distinct serotypes. Recombinant nucleocapsid polypeptide expressed in bacteria with a histidine tag at the amino terminus has been used as antigen for developing an assay to detect IBV-specific antibody. This fusion protein was produced readily in bacteria and easily purified with a nickel column which bound to the histidine tag. Conditions were optimized for using these preparations for an IBV-specific ELISA. Although differences in optical densities could be detected between pre-immune and positive sera for the Ark, Mass, and Gray strains with antigen concentrations between 50 and 0.1 microg per well, the greatest differences could be detected with 3 and 1.5 microg of protein per well. Maximum differences in optical densities between pre-immune and positive sera were obtained using 2.4 microg per well of protein and sera diluted between 1:80 and 1:160. In addition, as little as 30 ng/dot of recombinant nucleocapsid consistently detected IBV-specific sera in immunoblot assays which have convenient field applications.

The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection.

Specific cytotoxic T-lymphocyte (CTL) responses to nucleocapsid of infectious bronchitis virus (IBV) were identified by using target cells infected with a Semliki Forest virus (SFV) vector. Effector cells for CTL assays were collected from chickens infected with the Gray strain of IBV or inoculated with a DNA plasmid encoding nucleocapsid proteins. IBV-specific CTL epitopes were mapped within the carboxyl-terminal 120 amino acids of the nucleocapsid protein. CTL lysis of target cells infected with SFV encoding nucleocapsid was major histocompatibility complex restricted and mediated by CD8+ T cells. In addition, splenic T cells collected from chickens inoculated in the breast muscle with a DNA plasmid encoding this CTL epitope(s) recognized target cells infected with wild-type virus or an SFV vector encoding nucleocapsid proteins. CTL activity of splenic T cells collected from chicks immunized with a DNA plasmid encoding CTL epitopes was cross-reactive, in that lysis of target cells infected with serologically distinct strains of IBV was dose responsive in a manner similar to that for lysis of target cells infected with the homologous strain of IBV. Furthermore, chickens immunized with a DNA plasmid encoding a CTL epitope(s) were protected from acute viral infection.

Cellular mechanisms of feline immunodeficiency virus (FIV)-induced neuropathogenesis.

The high incidence of neurologic dysfunction from human immunodeficiency virus (HIV) infection has heightened interest in neuropathogenesis of other lentiviruses, including that associated with feline immunodeficiency virus (FIV). Both HIV and FIV efficiently enter the central nervous system and cause primary neurological disease that is not attributable to opportunistic infections or systemic disease. Cells in the brain infected by FIV are similar to those observed in HIV infection, both viruses infect macrophages, microglia, and astrocytes. Although substantial neuronal loss can occur in the cortex of HIV- or FIV-infected patients, most studies agree that neurons are not infected and indirect mechanisms of neurotoxicity are postulated. This review describes recent information on the neuropathogenesis of FIV and how this information correlates with what is known about the neuropathogenesis of HIV. Although the pathogenesis of neurological dysfunction in HIV- and FIV-infected patients is far from clear, it is becoming increasingly evident that the relationship between lentivirus presence in the brain and neurological signs is not straightforward and cannot be explained by simple cytolytic infection. The observed neurologic dysfunction is likely multifactorial and complex involving an intricate web of subcellular pathways and neurotoxic factors interacting with multiple cell types.

Specific cytotoxic T lymphocytes are involved in in vivo clearance of infectious bronchitis virus.

Cytotoxic T-lymphocyte (CTL) responses to infectious bronchitis virus (IBV) were determined at regular intervals between 3 and 30 days postinfection (p.i.). The maximum response with 82% lysis of labeled target cells was detected at 10 days p.i. The specific CTL response did not begin to decline until the amount of virus, which peaked at day 8 p.i. in both the kidneys and lungs, started to decrease. Clinical respiratory signs of illness also correlated with amount of virus. CTL activity was shown to be major histocompatibility complex (MHC) class restricted because the lysis of MHC-mismatched targets was negligible, and lysis was mediated by CD8+ CD4- T cells, as the CTL response could be abolished with removal of CD8+ CD4- but not CD4+ CD8- lymphocytes. In contrast, immunoglobulin M (IgM) antibody was not detected until day 10 p.i., and levels peaked at day 12 p.i.; IgG antibody levels were minimal until day 15 p.i. but continued to increase exponentially until day 30 p.i., the last day examined. In summary, CTL responses correlated with initial decreases in infection and illness.

Experimental confirmation of recombination upstream of the S1 hypervariable region of infectious bronchitis virus.

Chimeric infectious bronchitis virus (IBV) genomes with cross-over sites in the S1 gene were generated by co-infection with two distinct IBV strains. Recombinant viruses were collected from chicken embryos, embryonic cultured cells and chickens co-infected with Ark99 and Mass41 strains and purified by differential centrifugation. The recombinant S1 genes were identified by reverse transcription polymerase chain reaction (RTPCR) using heterologous primers and confirmed by nucleotide sequencing. The recombinants with Ark99 5' and Mass41 3' sequences were identified following the in vitro, in ovo and in vivo co-infections. Mixed RNA extracted from Ark99 and Mass41 did not produce RTPCR products with these primers at the PCR conditions used. Cross-over sites within the amplified 580 (Mass41) or 604 (Ark99) bases of the 5' S1 gene could only be detected between nucleotides 50 and 155. While this region, lying upstream of the S1 hypervariable region, corresponded with sites commonly identified in naturally occurring isolates, recombination sites identified in these studies could not be detected within the HVR of S1 of the genomes of chimeric viruses.

Ovine fetal malformations induced by in utero inoculation with Main Drain, San Angelo, and LaCrosse viruses.

The teratogenic potential of three bunyaviruses, two California serogroup bunyaviruses, LaCrosse virus and San Angelo virus, and a Bunyamwera serogroup member, Main Drain virus, in sheep was studied following in utero inoculation of ewes in early gestation. Although Main Drain virus appeared to be most teratogenic, all three viruses induced a range of lesions including arthrogryposis, hydrocephalus, fetal death, axial skeletal deviations, anasarca, and oligohydramnios. The teratogenic effects of these viruses are identical to those described in ovine infections by Cache Valley and Akabane viruses. Demonstration of a common bunyaviral tropism for fetal tissue infection that results in congenital brain and musculoskeletal malformations provides evidence that human in utero infection by bunyaviruses could result in similar malformations in human infants.

The infectious bronchitis virus nucleocapsid protein binds RNA sequences in the 3' terminus of the genome.

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a bacterial fusion protein which differed from the native protein only in the addition of six amino terminus histidine residues. Using RNA overlay protein blot assays, the recombinant protein was shown to bind to RNA fragments specific for the positive sense 3' noncoding end of the IBV genome. At greater concentrations of sodium chloride, the native and fusion nucleocapsid proteins similarly bound to G RNA, representing the terminal 1805 3' nt of the genome, whereas bovine serum albumin and allantoic fluid protein did not bind to labeled G RNA. Competitive gel shift assays with labeled G RNA indicated that the protein interacted with several unlabeled RNA representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the small segment cDNA also inhibited the interaction with IBV G RNA to approximately the same extent as homologous unlabeled G RNA, whereas reactions with bovine liver RNA and yeast tRNA were considerably weaker. Whereas yeast tRNA did not inhibit the interaction with the labeled large G RNA, interactions of the fusion protein with EF, a region from 78 to 217 nt from the 3' terminus of the IBV genome, were also apparently weaker than interactions with fragment CD which consisted of the 3' terminal 155 nt. On a molar basis, the latter interacted in an identical nature to a RNA consisting of CD and an additional 1053 nt of plasmid sequences. Compared to bovine liver RNA, unlabeled G specifically inhibited binding to the two smaller labeled IBV fragments in gel shift assays. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present on the genome, and may represent a common mechanism used by similar viral nucleoproteins whose functions depend on binding to RNA.

Retroviral vector-transduced cells expressing the core polyprotein induce feline immunodeficiency virus-specific cytotoxic T-lymphocytes from infected cats.

The core polyprotein of feline immunodeficiency virus (FIV) was expressed in primary feline T-lymphocytes using a retroviral vector. These cells were used as antigen-presenting stimulator cells (APSC) for the in vitro induction of cytotoxic T-lymphocytes (CTL) from feline peripheral blood mononuclear cells (PBMC). CTL from 4 cats chronically infected with the Petaluma strain of FIV specifically lysed autologous FIV-infected targets in an MHC-restricted manner. The CD8 phenotype of more than 70% of the induced effector cells (97% for cells from one cat) was consistent with MHC class I-restricted cytotoxicity. In addition, it was possible to detect low levels of core polyprotein-specific lysis from effector cells of two of the FIV-infected cats. When observed, the level of lysis, measured as a percentage of specific 111In release, was lower for the transgenic gag-expressing targets than for FIV-infected targets. The difference in killing may reflect the low level of core CTL were not detected in either PBMC stimulated with cells transduced by a retroviral vector without the FIV gag sequence or PBMC from an uninfected cat stimulated with autologous transgenic APSC. The detection of FIV-specific CTL from infected cats following stimulation with transgenic APSC suggests a role for retroviral vectors in determining CTL specific for individual lentiviral proteins in protective immunity.

Feline immunodeficiency virus (FIV)-specific cytotoxic T lymphocytes from chronically infected cats are induced in vitro by retroviral vector-transduced feline T cells expressing the FIV capsid protein.

We have previously reported the presence of feline immunodeficiency virus (FIV)-specific, major histocompatibility complex (MHC)-restricted cytolytic T lymphocytes (CTL) in experimentally FIV-infected cats. However, the fine specificity of the CTL and the role of individual FIV proteins in inducing FIV-specific CTL responses remain unknown. In this study, we examined the in vitro induction and activity of FIV p24 capsid-specific CTL obtained from cats that had been experimentally infected with FIV Petaluma for 30 to 56 months. An amphotropic murine retroviral vector was used to generate transgenic primary feline T lymphoblasts that expressed the FIV capsid protein. When the autologous capsid-transduced T cells were used in vitro to stimulate CTL responses from peripheral blood mononuclear cells of chronically infected cats, MHC-restricted lysis of virus-infected target cells was observed. The majority of the CTL expressed CD8, and depletion of this population, but not CD4+ cells, effectively diminished the CTL activity. When the autologous capsid-transduced T cells were used as target cells, lysis by capsid-induced effectors was not observed. Analysis of capsid-transduced T cell clones revealed a variable and low level of capsid expression among the clones. This study demonstrates the potential for using retroviral vectors as a means of inducing CTL effector cells that will specifically kill lentivirus-infected cells during lentiviral infection.