Identification and localization of conserved antigenic epitopes on the G2 proteins of California serogroup Bunyaviruses.

California (CAL) serogroup Bunyaviruses are significant agents of arboviral encephalitis in humans. They are maintained and transmitted in nature by mosquitoes to preferred vertebrate amplifying hosts. The G2 envelope glycoprotein of La Crosse virus (LAC) was proposed by Ludwig et al. to be a determinant for virus attachment to mosquito midgut cells. Monoclonal antibodies to G2 neutralize the infectivity of pronase-treated virus for mosquito cells. We determined the location of antigenic sites on the LAC G2. We showed that antigenic areas present on the LAC G2 protein are conserved among viruses in the California encephalitis and Melao subgroups of the CAL serogroup, but not in trivatattus virus, nor within the BUN serogroup. A comparison of the G2 exodomain amino acid sequences of eight CAL and three BUN viruses with monoclonal antibodies (MAb) binding data predicted the possible location of the antigenic sites. We used in vitro mutagenesis of the LAC G2 gene to construct a set of G2 genes with replacement sequences in the coding regions for the suspected MAb binding sites. The native and mutated proteins were expressed in Hela cells and the ability of MAbs to bind to the expressed proteins was tested. Four discontinuous amino acid sequences, conserved among eight CAL serogroup viruses, were identified as contributing to two conformational binding domains for neutralizing LAC G2 MAbs.

Potential for evolution of California serogroup bunyaviruses by genome reassortment in Aedes albopictus.

Aedes albopictus was introduced into the United States in used tires in 1985. Its successful colonization of the upper Midwest has potential to alter the current epidemiology of bunyaviruses that circulate in the region. It is permissive for the replication of several arboviruses, including La Crosse (LACV) and Jamestown Canyon (JCV) bunyaviruses. In this study, we demonstrate the ability of LACV and JCV to coinfect Ae. albopictus mosquitoes and to form all six possible reassortant genotypes. All reassortant viruses infect Ae. albopictus orally and can be transmitted to suckling mice. All reassortants are neurovirulent in mice. However, reassortant viruses carrying the LACV M segment in the foreign genetic background of JCV are more neuroinvasive than JCV, or any other reassortant genotype. In addition, these reassortants can replicate in gerbils and infect Ae. triseriatus, characteristics of LACV, but not JCV. Because Ae. albopictus is spreading into new geographic areas and feeds on a variety of mammals, including humans, it has the potential to transmit new, emerging bunyaviruses in nature.

Induction and maintenance of gastric ulceration in horses in simulated race training.

Gastric ulceration is a prevalent condition of racehorses. A number of models of gastric ulceration have been described, but none mimic the conditions of a horse in training. The objectives of this study were to determine whether gastric ulcers could be induced and maintained in a group of horses in simulated race training. In addition, serum cortisol was measured on a weekly basis to investigate the possibility that stress may be important in the pathogenesis of gastric ulceration. Thirty horses used in the trial were fed Bermuda grass hay and 6 kg of a concentrate diet, and exercised 6 days/week at speed over a distance of 1.6-2.4 km. Serum was collected and gastroendoscopic examinations performed on a weekly basis for the duration of the trial. All horses developed moderate to severe ulceration, and ulcers were maintained for the 56 day period of the trial. Only one horse had signs of abdominal discomfort, which resolved with minimal symptomatic treatment and without the use of anti-ulcer medications. Serum cortisol remained within reference ranges for the duration of the trial. Although there was some variation between the weekly examinations, serum cortisol concentrations were decreased from values obtained at the start of the trial. In this study ulcers developed without the administration of nonsteroidal anti-inflammatory agents or withholding of feed. This model provides a method to study the condition, and to investigate the effects of medications on the healing of ulcers in racehorses.

Lack of detectable interaction between HSV-1 and integrins or tachykinins.

Several viruses are known to utilize cellular integrin molecules to gain entry into cells. Because of the ability of herpes simplex virus type 1 (HSV-1) to disrupt cellular adhesion, as seen particularly in ocular infections, we examined the ability of several peptides, containing known integrin recognition sequences, to interfere with plaque formation of HSV-1 in epithelial cells. We also examined the possible involvement of tachykinins in virus entry. We did not detect any decrease in plaque formation by HSV-1 in the presence of Arg-Gly-Asp, Asp-Gly-Glu-Ala, or EILDV peptides or in the presence of monoclonal antibodies to the human beta1 or beta4 integrin subunit. Substance P or inhibitors of the NK1 or NK2 tachykinin receptors also had no inhibitory effects on HSV-1 plaque formation.

Sequence and cognitive analyses of two virulence-associated markers of bluetongue virus serotype 17.

Genome segments 2 and 3 were completely sequenced for one virulent and one avirulent bluetongue serotype 17 (BLU-17). These two segments were previously shown to exhibit virulence-associated markers. The marker on segment 2 was characterized as a change in the neutralization domain on its protein product, VP2. The nucleotide sequences for segments 2 were 94.5% identical, and their predicted proteins differed by 34 amino acids or 3.7%. Three clusters of variability were identified which may be involved with viral neutralization. These variable regions were compared to mutations for published monoclonal antibody-resistant variants of BLU. The marker on segment 3 was characterized as a mobility shift in polyacrylamide gel electrophoresis (PAGE). The nucleotide sequences were 95.0% identical, and their predicted proteins differed by four amino acids or 0.4%. These amino acid changes were relatively conserved; therefore, they are not likely responsible for virulence. The segment 3 sequences were compared to published sequences, and evidence was found to suggest that the virulent isolate had naturally reassorted between a BLU-17 and BLU-10 isolate.

A complex neutralization domain of bluetongue virus serotype 17 defines a virulence-associated marker.

A panel of seven monoclonal antibodies (MAb) was used to characterize a virulence-associated marker on bluetongue virus serotype 17 (BLU-17). These MAbs poorly neutralize virulent BLU-17 isolates, but effectively neutralize avirulent isolates (2). The MAbs immunoprecipitated VP2, an outer capsid protein, of both virulent and avirulent BLU-17 isolates despite their failure to neutralize the virulent isolates. The molecular mass (M(r)) of VP2 was calculated from the mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The M(r) of VP2 was estimated as 100,000 Da for the virulent isolates and 97,500 Da for the avirulent isolates. The seven MAbs were tested in a competitive enzyme-linked immunosorbent assay (ELISA) and found to bind at least three overlapping epitopes. In addition, neutralization-resistant variants were selected for five different MAbs. The Variants were tested in virus neutralization assays against the panel of seven MAbs, and three major neutralization patterns were observed, again suggesting at least three distinct epitopes. Minor differences within each neutralization pattern were also observed. The results from the binding and neutralization studies suggested that the seven MAbs define a complex neutralization domain on VP2, comprising at least three overlapping epitopes.

An analysis of co-circulating serotypes for bluetongue-17 virulence markers.

We have recently identified two markers associated with virulent strains of bluetongue virus serotype 17. These differences are an altered antigenic structure of the outer capsid protein VP2 and an increased electrophoretic mobility of the RNA segment 3 that codes for an inner core protein. We did not observe these markers in confirmed avirulent strains of bluetongue virus serotype 17. We hypothesized that these virulence-associated markers may have been acquired by bluetongue-17 through genetic interaction with other circulating serotypes of the virus. To test this hypothesis, we studied all isolates of other BLU serotypes obtained from the same sentinel cattle herds in Central America and the Caribbean on the same days as BLU-17 isolates. We looked for evidence of common epitopes on VP2 or an RNA segment 3 of identical mobility to that of the virulent strains of BLU-17. We found no evidence to indicate that genetic interaction with other co-circulating serotypes gave rise to these two specific virulence-associated markers of BLU-17.

Selective amplification of simian immunodeficiency virus genotypes after intrarectal inoculation of rhesus monkeys.

Animal models for sexual transmission of human immunodeficiency virus can define the influences of virus type, dose, and route of inoculation on infection and clinical outcome. We used an uncloned simian immunodeficiency virus stock (SIVmac) to inoculate cells in vitro and to inoculate rhesus monkeys by intravenous and intrarectal routes. The distribution of virus genotypes present in each of these infection examples was characterized by DNA sequence analysis of viral long terminal repeats (LTRs). Our analysis of LTR sequences from in vitro and in vivo infections revealed three main genotypes: one genotype was observed only for in vitro infection, and two other genotypes were recovered only from infected animals. By comparing animals inoculated with high intrarectal doses of SIVmac and those inoculated with low doses, we demonstrated that unique subsets of the stock were selected after intrarectal infection. Our findings indicate that minor genotypes present in the stock cross the rectal mucosa and are amplified selectively to become prominent in peripheral blood mononuclear cells from acutely infected animals. Studies with a molecular recombinant of SIV and human immunodeficiency virus type 1 sequences, SHIV, showed that viral LTR sequences do not undergo especially rapid sequence variation or rearrangement after intrarectal inoculation. The mucosal barrier exerts a significant influence on infection and disease progression by reducing the efficiency of SIVmac infection and by permitting distinct, pathogenic genotypes to become established in the host.

Antihistone antibody profile in sulfasalazine induced lupus.

Two patients developed drug induced lupus secondary to sulfasalazine (SSZ). One patient was receiving SSZ for Crohn's disease and was subsequently treated with olsalazine, which lacks the sulfapyridine component of SSZ. Her inflammatory bowel disease (IBD) remained controlled and she did not develop a recurrence of lupus, suggesting that olsalazine is safe in patients with IBD and a history of SSZ induced lupus. The SSZ induced antibodies were predominantly IgG against the (H2A-H2B)-DNA complex. Since lupus induced by 7 other drugs was associated with a similar antibody response, our findings support the existence of a common pathway for autoantibody induction.

Antistaphylococcal antibodies in dogs with recurrent staphylococcal pyoderma.

Staphylococcus intermedius skin infection (pyoderma) may be perpetuated in some dogs by a hypersensitivity reaction to staphylococcal organisms. Dogs with idiopathic superficial or deep recurrent staphylococcal skin infections may thus have quantitative differences in serum antistaphylococcal IgE antibodies compared with healthy dogs. To test this hypothesis, antistaphylococcal IgG and IgE antibodies were measured by ELISA in groups of dogs with idiopathic recurrent pyoderma, recurrent pyoderma secondary to atopic disease, non-recurrent pyoderma, and in healthy dogs. All groups of dogs with prior staphylococcal skin infection had significantly higher mean serum antistaphylococcal IgG levels than healthy dogs (P < 0.05). Dogs with recurrent deep pyoderma had the highest mean levels of antistaphylococcal IgG. Dogs with idiopathic recurrent superficial pyoderma and those with recurrent pyoderma secondary to atopy had significantly (P < 0.05) higher mean levels of serum antistaphylococcal IgE than other groups tested. It is concluded from these findings that S. intermedius can behave as an allergen in some dogs and elicit an IgE response. These results support the concept that bacterial hypersensitivity may be responsible for initiating or perpetuating skin lesions in these animals.

Virulence-associated antigenic and genetic characteristics of bluetongue virus-17 isolates.

Bluetongue virus (BLU), an orbivirus, is of importance to the sheep and cattle industries. We have obtained 5 United States BLU-17 isolates which have been tested for virulence in sheep and 16 BLU-17 field isolates from the Caribbean and Central America. Using a panel of 15 monoclonal antibodies (MAb) against an avirulent BLU-17, we observed that 6 MAbs had negligible or very low neutralization titers for the virulent isolates in contrast to moderate to high titers for the avirulent isolates. These MAbs also differentiated the field isolates into two groups--inadequate vs effective neutralization. All 6 MAbs immunoprecipitated the outer capsid protein, VP2. Electropherotyping of genomic RNA from all 21 viruses identified an increase in RNA segment 3 mobility for those isolates which were not neutralized by the 6 specific MAbs. RNA segment 3 codes for the inner core protein, VP3. There were no detectable electrophoretic differences for RNA segment 2, which encodes VP2. In summary, the virulent BLU-17 isolates differed from the avirulent isolates in both the antigenicity of the outer capsid protein, VP2, and the electrophoretic mobility of RNA segment 3, and we hypothesize that one or both of these changes may result in BLU virulence.

Migraine as a sequela to chronic low back pain.

The occurrence of headache as a sequela of low back pain was examined in a sample of chronic pain patients. All patients had low back pain without history of head, neck, or upper back injury or headache onset simultaneous with the low back pain. Consistent with prior research, headache was found to be a common concomitant of back pain. In many patients, headache was found to have begun or exacerbated markedly after onset of low back pain. Prevalence of migraine in female patients was significantly higher than the population prevalence for females in the United States; this was not true for male patients. Potential mechanisms for explaining the high prevalence of migraine following low back pain are discussed, including increased muscle tension, psychosocial factors, and analgesic overuse.

Cellular immune responses in rhesus macaques infected rectally with low dose simian immunodeficiency virus.

Monkeys infected rectally with low dose simian immunodeficiency virus (SIV) were resistant to high dose challenge with SIV. Peripheral blood mononuclear cells (PBMC) from two of four challenged monkeys were unable to support SIV replication in vitro unless cultures were depleted of CD8+ lymphocytes. Monkeys that had survived high dose rectal infection with SIV also suppressed virus replication in cultured PBMC. PBMC from uninfected monkeys supported virus replication in both unfractionated and CD8-depleted cultures. Virus-suppressive activity of PBMC may be an important correlate of protective immunity in AIDS.

Primary cultures of rhesus placental syncytiotrophoblasts are permissive for SIV infection.

Primary cultures of rhesus syncytiotrophoblasts incubated with SIVdeltaB670, SIVmac251, or SIVmac239 produced readily detectable virus in the supernatant for up to three weeks after infection. At four weeks, cells generally failed to release virus but placental cell lysates and placental cells cocultured for 24 hours with uninfected CEM x 174 cells were able to transmit infection. The presence of virus was confirmed by electron microscopy and PCR amplification of viral sequences from trophoblast genomic DNA. SIV p27 antigen was localized by immunostaining primarily in syncytiotrophoblasts.

Production and characterization of mouse monoclonal antibodies directed against canine IgE and IgG.

Immediate-type hypersensitivity reactions have been studied infrequently in dogs, in part because of limited availability of antisera specific for canine IgE. A series of murine hybridoma cell lines were prepared, that produced monoclonal antibodies (MAb) with specificity for canine immunoglobulin E (IgE) and IgG. The MAb were tested for their ability to induce a reverse cutaneous anaphylaxis reaction in dog skin, to neutralize the Prausnitz-Küstner reactivity of atopic dog serum, to serve as a ligand in immunoaffinity chromatography, and to bind to IgE and other Ig subclasses in several ELISA systems. Some of the MAb produced were found to be specific for canine IgE. Other MAb recognized common or similar determinants on IgE and IgG, or on IgG and IgM, though with apparently differing affinities. Heat or acid treatment of canine IgE abolished most, but not all, of the reactivity with the anti-IgE MAb. These MAb will be useful for further study of IgE-mediated phenomena in the dog.

Pathogenesis of SIVmac251 after atraumatic inoculation of the rectal mucosa in rhesus monkeys.

Intrarectal inoculation of rhesus monkeys with low doses of SIVmac led to a prolonged clinical and virological latency that was not observed for high intrarectal doses or for intravenous inoculation. Animals infected intrarectally with low virus doses remained negative for serum antibody responses to SIV for at least one year even though they readily transferred SIV to naive recipients via transfusion of whole blood.

Muramyl peptides augment the in vitro and in vivo cytostatic activity of canine plastic-adherent mononuclear cells against canine osteosarcoma cells.

A tumor cytostasis assay was developed that measured the effect of the immunomodulator muramyl dipeptide (MDP) on the in vitro cytostatic activity of canine plastic-adherent mononuclear cells. Mononuclear cells were isolated from the peripheral blood of healthy Beagle donors and allowed to adhere to a 96-well microtiter plate. The adherent cell population was characterized by cell morphology, non-specific esterase staining, and flow microfluorometry to be approximately 42% monocytes, 49% lymphocytes, and 8% eosinophils. Canine plastic-adherent mononuclear cells spontaneously caused cytostasis of D-17 canine osteosarcoma target cell proliferation. The spontaneous cytostatic activity of adherent mononuclear cells was significantly augmented by exposure to MDP or to lipopolysaccharide (LPS), with maximal cytostatic activity being observed after combined exposure to MDP and LPS. Mononuclear cell cytostasis toward D-17 canine osteosarcoma and A375 human melanoma cells was enhanced (P < 0.05) when normal dogs were administered liposome-encapsulated muramyl tripeptide phosphatidylethanolamine, a lipophilic derivative of MDP, by intravenous injection.

Monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus.

Neutralizing monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus (LACV) were prepared. Two antibodies immunoprecipitated the 120 kDa virus attachment protein for vertebrate cells, G1, while five immunoprecipitated the 35 kDa G2 protein, whose function is currently unknown. Two monoclonal antibodies were obtained that specifically precipitated both G1 and G2 from [35S]cysteine labeled LACV infected cell lysates. The G2 specific monoclonal antibodies had high neutralizing titers when assayed in mosquito cells but limited ability to neutralize virus in mammalian cells. The G1/G2 specific antibodies neutralized virus infectivity in both vertebrate and invertebrate cells at high titers. These results suggest that G2 is involved in the interaction of virus with mosquito cells and that G1 and G2 may share a common structural epitope relevant to their role as attachment proteins in vertebrate and mosquito cells. Monoclonal antibodies directed against G2 or G1/G2 have not previously been reported and should be useful tools for characterizing the biological functions of these molecules in the divergent micro-environments of vertebrate and invertebrate hosts.

Role of La Crosse virus glycoproteins in attachment of virus to host cells.

Data presented in this report demonstrate that the initial event of La Crosse virus (LACV) infection of cells is probably the interaction of viral glycoproteins with specific cellular receptor sites. We have shown that LACV glycoprotein G1 binds, in a dose-dependent manner, to continuous vertebrate and mosquito cell lines, but not to mosquito midguts isolated ex vivo. This binding can be inhibited by the pretreatment of cells with excess homologous glycoprotein but not with excess heterologous LACV glycoprotein. In contrast, we have shown that LACV glycoprotein G2 binds to the continuous mosquito cell line and vector midgut cells, but not to vertebrate cells. LACV infection of vertebrate cells can be inhibited by treatment of cells with purified G1, while infection in mosquito cells can be reduced by treatment of cells with a combination of G1 and G2. The results suggest that G1 is the viral attachment protein (VAP) for vertebrate cells, and that G2 serves the same purpose for mosquito midgut cells. We speculate that the protease-resistant G2 molecule may have evolved to serve as the VAP in the midgut under conditions in which G1 might be altered or removed from the virus envelope, and thus is essential to the evolution and maintenance of vertebrate-invertebrate transmission cycles.

Effects of thromboxane synthetase inhibition on immune complex glomerulonephritis.

To determine the role of thromboxane A2 in the pathogenesis of experimentally induced immune complex glomerulonephritis, 12 concanavalin A-immunized Beagles were infused with 1 mg of concanavalin A via each renal artery and treated twice daily for 8 days with either 30 mg of CGS 12970/kg, PO, a specific thromboxane synthetase inhibitor, or placebo. The effect of treatment was assessed by measuring endogenous creatinine clearance and urine protein and eicosanoid excretion, and by evaluating changes in glomerular morphometric characteristics. On postinfusion day 8, urine protein, thromboxane B2, and 11-dehydro-thromboxane B2 excretion, glomerular epithelial crescent formation, and glomerular cell proliferation in the CGS 12970-treated dogs were significantly decreased when compared with values in the placebo-treated group. Differences were not observed in endogenous creatinine clearance, urine prostaglandin E2 and 6-keto-prostaglandin F1 alpha excretion, or glomerular polymorphonuclear leukocyte infiltration between groups in this study. These findings suggest thromboxane A2 has a role in the development of immune complex glomerulonephritis and that thromboxane synthetase inhibition may be beneficial in attenuating some of the functional and histological changes associated with immune complex glomerulonephritis.