The Fate of Ornamental White Fringetree Through the Invasion Wave of Emerald Ash Borer and Implications for Novel Host Use by This Beetle.

Emerald ash borer (EAB, Agrilus planipennis Fairmaire [Coleoptera: Buprestidae]) is a wood boring beetle that is an invasive pest of ash trees (Fraxinus spp.) in North America. In 2014, it was reported that EAB had infested white fringetree (Chionanthus virginicus L. [Lamiales: Oleaceae]) in Ohio and was since found to have infested this species across its invasive range. In 2018, we reexamined 166 white fringetrees in Illinois, Indiana, Ohio, and Pennsylvania that had been previously examined for EAB attack in 2015 to determine their fate. We assessed tree health and EAB infestation in each tree, assigned an infestation status of newly, continuously, not reinfested, or never infested, and compared the trees' current status to their 2015 status. This assessment was done to determine whether their health and infestation status had changed through the EAB invasion wave. We found that attack rates declined: 26% of trees were infested in 2015 whereas only 13% were in 2018, likely coinciding with declining beetle populations in the area. Overall tree health improved for trees that were not reinfested by EAB after a record of attack in 2015, suggesting that they can survive and recover from EAB attack. Conversely, health declined for newly and continuously infested trees, indicating that they became stressed from EAB attack. Although the majority of the trees survived the invasion wave, several were removed from various sites due to EAB attack suggesting that white fringetree varies in its resistance and tolerance to attack. As beetle populations continue to expand geographically, infestation rates will likely increase and health of white fringetrees will decrease with the EAB attack wave, especially as EAB reaches denser populations of fringetrees.

Designing a therapeutic hepatitis B vaccine to circumvent immune tolerance.

An effective prophylactic hepatitis B virus (HBV) vaccine has long been available but is ineffective for chronic infection. The primary cause of chronic hepatitis B (CHB) and greatest impediment for a therapeutic vaccine is the direct and indirect effects of immune tolerance to HBV antigens. The resulting defective CD4+/CD8+ T cell response, poor cytokine production, insufficient neutralizing antibody (nAb) and poor response to HBsAg vaccination characterize CHB infection. The objective of this study was to develop virus-like-particles (VLPs) that elicit nAb to prevent viral spread and prime CD4+/CD8+ T cells to eradicate intracellular HBV. Eight neutralizing B cell epitopes from the envelope PreS1 region were consolidated onto a species-variant of the HBV core protein, the woodchuck hepatitis core antigen (WHcAg). PreS1-specific B cell epitopes were chosen because of preferential expression on HBV virions. Because WHcAg and HBcAg are not crossreactive at the B cell level and only partially cross-reactive at the CD4+/CD8+ T cell level, CD4+ T cells specific for WHcAg-unique T cell sites can provide cognate T-B cell help for anti-PreS1 Ab production that is not curtailed by immune tolerance. Immunization of immune tolerant HBV transgenic (Tg) mice with PreS1-WHc VLPs elicited levels of high titer anti-PreS1 nAbs equivalent to wildtype mice. Passive transfer of PreS1 nAbs into human-liver chimeric mice prevented acute infection and cleared serum HBV from mice previously infected with HBV in a model of CHB. At the T cell level, PreS1-WHc VLPs and hybrid WHcAg/HBcAg DNA immunogens elicited HBcAg-specific CD4+ Th and CD8+ CTL responses.

Immunological features in pediatric patients with recurrent and severe infection: Identification of Primary Immunodeficiency Diseases in Merida, Venezuela.

INTRODUCTION AND OBJECTIVES: Primary immunodeficiency diseases (PIDs) are disorders associated mainly with recurrent and severe infection and an increase in susceptibility to autoimmune conditions and cancer. In Venezuela, PIDs are underdiagnosed and there is usually a delay in their diagnosis. Hence there are no data concerning the frequency and type of PIDs that occur. The aim of this study was to identify and quantify the types of PIDs that occur in Merida, a population within Venezuela. PATIENTS OR MATERIALS AND METHODS: Following an informative program designed to alert local health professionals to the warning signs for PIDs, patients with a history of recurrent infections were referred to the Instituto de Inmunologia Clinica, Universidad de Los Andes. RESULTS AND CONCLUSIONS: During the three-year period January 2014 to January 2017, thirty-two cases of PIDs were identified in pediatric patients, and 17 different types of PIDs, were identified. Predominantly antibody deficiencies were most frequent (40.6%), followed by immunodeficiencies affecting cellular and humoral immunity (21.8%), congenital defects of phagocyte (18.7%), CID with associated or syndromic features (9.3%), defects in intrinsic and innate immunity (6.4%) and diseases of immune dysregulation (3.2%). These results have important implications not only to the future approach for management of patients in our regions, but add important knowledge concerning PIDs in Latin America and worldwide.

T-cell profiles elicited by Toxoplasma gondii in acutely/chronically infected humans.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that can infect almost all warm-blooded species and induce a chronic infection in human hosts. The aim of this work was to investigate Th1, Th2, Th17 and Treg polarization, induced by four important T. gondii antigens (SAG1, ROP1, GRA8 and MAG1) in acutely and chronically infected patients. For this purpose, SAG1, ROP1, GRA8 and MAG1 were expressed as recombinant proteins, purified, and used to evaluate the proinflammatory and regulatory immune response profiles in seropositive and seronegative individuals. Our results show that SAG1 and ROP1 elicited a proinflammatory profile (INF-γ, IL-12 and IL-17) in individuals in the acute phase, whereas MAG1 and GRA8 induced a regulatory pattern (Treg and TGF-ß) in chronically infected patients. These results reveal fundamental differences in T-cell polarization induced by T. gondii antigens, which could have important implications in the immunopathogenesis of the disease and in future proposals of therapeutic strategies.

Immune network analysis of cerebrospinal fluid in myalgic encephalomyelitis/chronic fatigue syndrome with atypical and classical presentations.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a persistent and debilitating disorder marked by cognitive and sensory dysfunction and unexplained physical fatigue. Classically, cases present after a prodrome consistent with infection; however, some cases are atypical and have a different presentation and comorbidities that pose challenges for differential diagnosis. We analyzed cerebrospinal fluid (CSF) from 32 cases with classical ME/CFS and 27 cases with atypical ME/CFS using a 51-plex cytokine assay. Atypical subjects differed in cytokine profiles from classical subjects. In logistic regression models incorporating immune molecules that were identified as potential predictor variables through feature selection, we found strong associations between the atypical ME/CFS phenotype and lower CSF levels of the inflammatory mediators, interleukin 17A and CXCL9. Network analysis revealed an absence of inverse inter-cytokine relationships in CSF from atypical patients, and more sparse positive intercorrelations, than classical subjects. Interleukin 1 receptor antagonist appeared to be a negative regulator in classical ME/CFS, with patterns suggestive of disturbances in interleukin 1 signaling and autoimmunity-type patterns of immune activation. Immune signatures in the central nervous system of ME/CFS patients with atypical features may be distinct from those with more typical clinical presentations.

Comparison of fin ray sampling methods on white sturgeon Acipenser transmontanus growth and swimming performance.

Effects of two fin-ray sampling methods on swimming performance, growth and survival were evaluated for hatchery-reared sub-adult white sturgeon Acipenser transmontanus. Fish were subjected to either a notch removal treatment in which a small section was removed from an anterior marginal pectoral-fin ray, or a full removal treatment in which an entire marginal pectoral-fin ray was removed. Control fish did not have fin rays removed, but they were subjected to a sham operation. A modified 3230 l Brett-type swim tunnel was used to evaluate 10 min critical station-holding speeds (SCSH ) of A. transmontanus, immediately after the fin ray biopsies were obtained with each method. Survival and growth were evaluated over a 6 month period for a separate group of fish subjected to the same biopsy methods. Mean ± S.E. 10 min SCSH were 108·0 ± 2·3, 110·0 ± 2·6 and 115·0 ± 3·5 cm s(-1) for the notch removal group, full removal group and control group, respectively, and were not significantly different among treatments. Behavioural characteristics including tail-beat frequency and time spent hunkering were also not significantly different among treatment groups swimming at the same speeds. There were no mortalities and relative growth was similar among treatment groups. Average biopsy time for the notch removal method was lower and the wounds appeared to heal more quickly compared with the full removal method.

Cytokine network analysis of cerebrospinal fluid in myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome is an unexplained debilitating disorder that is frequently associated with cognitive and motor dysfunction. We analyzed cerebrospinal fluid from 32 cases, 40 subjects with multiple sclerosis and 19 normal subjects frequency-matched for age and sex using a 51-plex cytokine assay. Group-specific differences were found for the majority of analytes with an increase in cases of CCL11 (eotaxin), a chemokine involved in eosinophil recruitment. Network analysis revealed an inverse relationship between interleukin 1 receptor antagonist and colony-stimulating factor 1, colony-stimulating factor 2 and interleukin 17F, without effects on interleukin 1α or interleukin 1ß, suggesting a disturbance in interleukin 1 signaling. Our results indicate a markedly disturbed immune signature in the cerebrospinal fluid of cases that is consistent with immune activation in the central nervous system, and a shift toward an allergic or T helper type-2 pattern associated with autoimmunity.

Cleavage of the IPS-1/Cardif/MAVS/VISA does not inhibit T cell-mediated elimination of hepatitis C virus non-structural 3/4A-expressing hepatocytes.

BACKGROUND: Hepatitis C virus (HCV) effectively establishes persistent infection in human livers. The non-structural (NS) 3/4A complex participates in this process by cleavage of interferon beta (IFN beta) promoter stimulator-1 (IPS-1; also termed Cardif/MAVS/VISA), which inhibits responses to double stranded (ds) RNA. However, it is not known whether this effect extends beyond innate responses. AIMS: To test if HCV NS3/4A affects innate and adaptive immune responses in vivo. METHODS: NS3 levels were semi-quantified in human liver biopsies, transfected cells, and in transgenic (Tg) mouse livers by western blot. The effect of NS3/4A on dsRNA-mediated signalling and on the integrity of IPS-1 was analysed using in vitro translation, transfected cells and Tg mice. Cytotoxic T cell (CTL)-mediated clearance of transient firefly luciferase (FLuc)- and/or NS3/4A-Tg hepatocytes was determined using in vivo imaging and western blot. RESULTS: NS3 protein levels were in a comparable range (0.1-49 microg/g tissue) in infected human livers and Tg mouse livers. Importantly, these levels of NS3/4A reduced murine innate responses to synthetic dsRNA in vivo, supporting the possibility that this occurs also in infected humans. The likely explanation for this was the NS3/4A-mediated cleavage of mouse IPS-1, albeit less efficiently than human IPS-1. Despite this, FLuc- and/or NS3/4A-expressing murine hepatocytes were effectively eliminated by hepatic CTLs, utilising the classical molecules for virus-infected cell lysis, including CD8, IFN gamma, perforin and FasL. CONCLUSIONS: Although HCV NS3/4A inhibits the innate immunity, this does not prevent CTL-mediated clearance of NS3/4A-expressing hepatocytes in vivo. Thus, other HCV proteins are most likely responsible for interfering with the adaptive immunity.

Wire initiation critical for radiation symmetry in z-pinch-driven dynamic hohlraums.

Axial symmetry in x-ray radiation of wire-array z pinches is important for the creation of dynamic hohlraums used to compress inertial-confinement-fusion capsules. We present the first evidence that this symmetry is directly correlated with the magnitude of the negative radial electric field along the wire surface. This field (in turn) is inferred to control the initial energy deposition into the wire cores, as well as any current shorting to the return conductor.

Experimental Bin Drenching System for Testing Biocontrol Agents to Control Postharvest Decay of Apples.

A portable drencher capable of drenching a single bin of fruit was built to simulate the commercial application of chemicals to harvested apples in small orchard operations in the central and eastern United States. The drencher required as little as 125 liters of the treatment solution and permitted various bin travel speeds. Wounded apples were placed midway between the bottom and top of the bin, in the center, and near the four corners of the bin (20 fruit per location) and covered with enough unwounded apples to fill the bin. The bins were drenched with a suspension containing Penicillium expansum at 2 × 104 conidia per ml in 2000, 5 × 103 conidia per ml in 2001, and 3 × 103 conidia per ml in 2002 and 2003. In 2000 and 2003, the additional treatments included a combination of P. expansum with the yeast Metschnikowia pulcherrima at ~;1.2 × 107 CFU/ml, and in 2003 a combination with 2% sodium bicarbonate (SB) or a mixture of the yeast and SB. After 3 months of storage at ~;2°C, at all P. expansum conidial concentrations, more than 90% of wounded fruit developed decay on 'Golden Delicious', 'Delicious', and 'Rome' apples in the 2000-02 experiments. In 2003, 66 and 33.1% of the wounded fruit developed decay on 'Delicious' and 'Golden Delicious', respectively. The application of the antagonist reduced decay to 39 and 3.3% on 'Golden Delicious' in 2000 and 2003, respectively, and to 26% on 'Delicious' in 2003. The addition of SB reduced decay on both cultivars and, in combination with the yeast, was the most effective treatment on 'Golden Delicious'. This portable drencher can be very useful for evaluating different treatments applied to apples after harvest at the commercial level.

Evaluation of initial base deficit as a prognosticator of outcome in the pediatric trauma population.

Although the utility of the base deficit as an indicator of hypoperfusion and physiologic derangement in adults is well established, its value in the assessment of children is not as clear. The purpose of this study was to evaluate this tool with regard to injury severity, infectious morbidity, and outcome in a pediatric trauma population. A retrospective review of a 6-year period of the database of our level 1 pediatric trauma center was performed. One hundred seventeen severely injured children requiring mechanical ventilation were identified. Initial base deficit, Injury Severity Score, time to correction of this abnormality, ventilator days, infectious morbidity, and mortality were obtained and compared. Of the 117 patients included in this study, 30 patients were identified with an initial BD of less than or equal to -8 mEq/L and were placed into group 1. Group 2 consisted of the remaining 87 patients who presented with a base deficit (BD) of greater than -8 mEq/L. An admission base deficit of -8 mEq/L or less corresponded to a probability of mortality of 23 per cent as opposed to only 6 per cent with a BD greater than -8. Patients in group 1 remained on mechanical ventilation 9.4 +/- 8.1 days, whereas patients in group 2 remained ventilated 6.5 +/- 6.4 days; an increase of nearly 145 per cent. Likewise, the number of infectious complications rose 26 per cent with a worsening initial base deficit from 17 per cent of group 2 patients to 43 per cent of group 1 patients. We conclude that a high initial base deficit in injured children predicts a higher incidence of infectious complications and a less favorable outcome. This readily available laboratory study can identify those children most at risk of potentially preventable complications.

Susceptibility of the Stem Pull Area of Mechanically Harvested Apples to Blue Mold Decay and Its Control with a Biocontrol Agent.

A new harvester, which uses a rapid displacement actuator on the main scaffolds to remove apples from trees with narrow-inclined trellises, has shown good potential. With this technique, stem loss (stempulls) during harvest ranges from 20 to 57%, depending on the cultivar. This can create a potential point of entry for pathogens. We evaluated the susceptibility of the stem cavity area, with and without stems, to blue mold decay (Penicillium expansum) on three cultivars of mechanically harvested apples, and tested the effectiveness of the antagonist Pseudomonas syringae (used in BioSave 110) in controlling decay. Fruit with stempulls were more susceptible to blue mold decay than fruit with stems. On fruit with stempulls inoculated with P. expansum and stored for 2 months at 1°C, decay incidence was 0% on 'Pink Lady', 8.3% on 'Ace Spur Delicious', and 41% on 'Empire'. On fruit with stems, there was no decay on all three cultivars. P. syringae reduced decay on 'Empire' with stempulls to 3.3%, and no decay occurred on the other two cultivars. Similar trends were observed on fruit stored at 22°C for 14 days, but the incidence of decay was higher, and only 'Pink Lady' had no decay on the antagonist-treated fruit. Although mechanical harvesting can predispose the stem cavity to decay in some cultivars, this problem can be alleviated using biological control without resorting to the use of synthetic pesticides.

Comparison of two methods for predicting aqueous solubility.

This study compares the solubility predictions of the two parameter general solubility equation (GSE) of Jain and Yalkowsky with the 171 parameter Klopman group contribution approach. Melting points and partition coefficients were obtained for each of the compounds from Klopman's test set. Using these two variables, the solubility of each compound was calculated by the GSE and compared to the values predicted by Klopman. Both methods give reasonable solubility predictions. The data of Klopman produced an average absolute error (AAE) of 0.71 and a root-mean-square error (RMSE) of 0.86, while the GSE had an AAE of 0.64 and a RMSE of 0.92.

Characterization of a novel hepatitis B virus mutant: demonstration of mutation-induced hepatitis B virus surface antigen group specific "a" determinant conformation change and its application in diagnostic assays.

We report and characterize a novel mutant of the hepatitis B virus surface antigen (HBsAg). The mutant was isolated from a symptomatic patient who was found to be persistently positive for both HBsAg and anti-hepatitis B surface antibody (anti-HBs) and a long-lasting anti-HBc (core) IgM. Due to the unusual immune serological profile, polymerase chain reaction and sequencing were performed and revealed a genotype D mutant HBV (LBN). Aligned with known HBsAg sequences from GenBank, the LBN variant matched to consensus subtype ayw2 and revealed five mutation positions. Interestingly, none of the mutations was found within the group-specific "a" determinant region (124-147) and, specifically, two of the five mutations, T118K and P120Q, were located only a few amino acids adjacent to the 124-147 region. Using a panel of six monoclonal antibodies and vaccine raised human neutralizing antibodies, the recombinant wild-type HBsAg and the novel variant LBN HBsAg were investigated for their immunological reactivity. Vaccine raised human anti-HBs showed less reactivity to the variant LBN HBsAg than to wild-type HBsAg in enzyme immune assays. Furthermore, our observations demonstrate the influence of adjacent residues on the group-specific "a" determinant structural configuration; this resulted in severe antigenic changes of the immunodominant region as well as in the subtype serology. The importance of the variant LBN lies in the observation that mutations close to the "a" determinant can change the immunodominant region structure and therefore alter the group specific determinant antigenicity even though no mutations are present within this region. Hence, the classical definition of the "a" determinant cluster may need to be extended to cover a broader region to include the requirement of adjacent amino acids to support its conformation. In conclusion, by understanding the HBsAg major immunodominant region structure and by using a combination of antibodies with specificity covering all key mutation locations, maximal anti-HBs-based protection, and highly sensitive diagnostic assays can be achieved.

Preclinical evaluation of group B streptococcal polysaccharide conjugate vaccines prepared with a modified diphtheria toxin and a recombinant duck hepatitis B core antigen.

An effective vaccine against group B streptococcal (GBS) disease will undoubtedly include capsular polysaccharides (CPSs) from each of the five serotypes prevalent in the United States individually coupled to immunogenic proteins. This formulation may require the use of two or more different protein carriers. We preclinically examined the potential of two proteins to serve as effective carriers for GBS type III CPS. Recombinant duck hepatitis B core antigen (rdHBcAg), a particulate protein of viral origin, and a newly mutated form of diphtheria toxin (DTm) were covalently and directly coupled to purified type III CPS by reductive amination. Seventy-seven of 79 (97%) newborn pups born to mouse dams actively vaccinated with type III CPS-rdHBcAg conjugate survived GBS type III challenge, whereas none of the pups born to dams that received an uncoupled mixture of type III CPS and rdHBcAg or saline survived. Likewise, 64 (98%) of 65 pups born to dams vaccinated with type III CPS-DTm conjugate survived challenge, in sharp contrast to no survivors among the pups born to dams vaccinated with an uncoupled mixture of type III CPS and DTm. The presence of type III CPS-specific IgG in serum from dams correlated with pup survival in groups that received a conjugate vaccine, and this serum was opsonically active in vitro against GBS type III. In addition, carrier-specific IgG was also measured in serum from vaccinated mice. These data suggest that the rdHBcAg and DTm may be effective carriers for GBS CPSs.

In vitro assembly of feline immunodeficiency virus capsid protein: biological role of conserved cysteines.

Core assembly, a key step in the retroviral life cycle, is poorly understood. Previous studies have shown that the entire gag region is needed to form the assembled particles. In this report, we have shown that the assembly process is driven by recombinant capsid protein (p26) of feline immunodeficiency virus itself. Proteins are expressed in a bacterial system and soluble forms of wild-type and modified proteins are purified from bacterial extracts and are examined on gel-filtration chromatography fitted to an HPLC system. It has also been shown that changing residue Cys190 (one of the two conserved cysteines of feline immunodeficiency virus which are also conserved for all the immunodeficiency viruses including HIV) to serine by site-directed mutagenesis disrupts the assembly process. In addition, this modification causes considerable thermal instability of the protein while substitutions at nonconserved cysteines do not significantly affect the thermal stability and assembly of the protein. These findings indicate that conserved cysteine residues play a vital role in the capsid protein assembly and, therefore, are critical for virus infectivity.

Molecular basis for the interaction of the hepatitis B virus core antigen with the surface immunoglobulin receptor on naive B cells.

The nucleocapsid of the hepatitis B virus (HBV) is composed of 180 to 240 copies of the HBV core (HBc) protein. HBc antigen (HBcAg) capsids are extremely immunogenic and can activate naive B cells by cross-linking their surface receptors. The molecular basis for the interaction between HBcAg and naive B cells is not known. The functionality of this activation was evidenced in that low concentrations of HBcAg, but not the nonparticulate homologue HBV envelope antigen (HBeAg), could prime naive B cells to produce anti-HBc in vitro with splenocytes from HBcAg- and HBeAg-specific T-cell receptor transgenic mice. The frequency of these HBcAg-binding B cells was estimated by both hybridoma techniques and flow cytometry (B7-2 induction and direct HBcAg binding) to be approximately 4 to 8% of the B cells in a naive spleen. Cloning and sequence analysis of the immunoglobulin heavy- and light-chain variable (VH and VL) domains of seven primary HBcAg-binding hybridomas revealed that six (86%) were related to the murine and human VH1 germ line gene families and one was related to the murine VH3 family. By using synthetic peptides spanning three VH1 sequences, one VH3 sequence, and one VLkappaV sequence, a linear motif in the framework region 1 (FR1)complementarity-determining region 1 (CDR1) junction of the VH1 sequence was identified that bound HBcAg. Interestingly, the HBcAg-binding motif was present in the VL domain of the HBcAg-binding VH3-encoded antibody. Finally, two monoclonal antibodies containing linear HBcAg-binding motifs blocked HBcAg presentation by purified naive B cells to purified HBcAg-primed CD4(+) T cells. Thus, the ability of HBcAg to bind and activate a high frequency of naive B cells seems to be mediated through a linear motif present in the FR1-CDR1 junction of the heavy or light chain of the B-cell surface receptor.

ELISPOT analysis of hepatitis C virus protein-specific IFN-gamma-producing peripheral blood lymphocytes in infected humans with and without cirrhosis.

An improved ability to monitor hepatitis C virus (HCV)-specific T cell immunity in infected patients may provide novel information regarding the pathogenesis and prognosis of this infection. We used an ELISPOT assay to analyze a cross-section of HCV-infected humans. HCV-infected patients without cirrhosis, those with cirrhosis, and controls with other liver diseases were tested for recall responses to HCV Core and NS3 proteins. Peripheral blood lymphocytes (PBLs) from HCV-infected patients without cirrhosis responded to NS3 and Core proteins, producing predominantly IFN-gamma, with little IL-4 or IL-5. In contrast, PBLs from HCV-infected patients with cirrhosis responded to NS3, but not to the Core protein, suggesting a selectively altered immune state during cirrhosis. Our data provide support for the notion that HCV-specific IFN-gamma-producing immunity is important in the pathogenesis of progressing HCV-related disease.

Use of passive ambient ozone (O3) samplers in vegetation effects assessment.

A stochastistic, Weibull probability model was developed and verified to simulate the underlying frequency distributions of hourly ozone (O3) concentrations (exposure dynamics) using the single, weekly mean values obtained from a passive (sodium nitrite absorbent) sampler. The simulation was based on the data derived from a co-located continuous monitor. Although at the moment the model output may be considered as being specific to the elevation and location of the study site, the results were extremely good. This effort for the approximation of the O3 exposure dynamics can be extended to other sites with similar data sets and in developing a generalized understanding of the stochastic O3 exposure-plant response relationships, conferring measurable benefits to the future use of passive O3 samplers, in the absence of continuous monitoring.

Urea equilibrium unfolding of the major core protein of the retrovirus feline immunodeficiency virus and its tryptophan mutants.

Circular dichroism and fluorescence spectroscopy have been employed to study the urea unfolding mechanism of a recombinant form of the major core protein of feline immunodeficiency virus (FIV-rp24) and its native tryptophan mutants. The equilibrium denaturation curves indicate the existence of two transitions. The first unfolding transition most likely reflects the denaturation of the carboxy-terminal region of FIV-rp24. Consequently, the second transition, where the changes in fluorescence are produced, should reflect the denaturation of the amino-terminal region. If the intermediate observed upon urea denaturation is an on-pathway species, the data described herein can reflect the sequential and independent loss of structure of the two domains that this type of proteins possesses.