Kevin T. Schultz

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.

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.

Enzyme processing of La Crosse virus glycoprotein G1: A bunyavirus-vector infection model

Efficient transmission, amplification, and dissemination of arboviruses require viral replication in vertebrate and invertebrate hosts. As a result, virions are exposed to two significantly different environments. Exposure of LaCrosse virus (LACV) to proteolytic enzymes, such as those that may be found in the mosquito midgut, increases virus affinity for mosquito cells. These enzymes remove the major envelope glycoprotein (G1) while leaving the second glycoprotein (G2) intact. Processing of LACV glycoproteins in the mosquito midgut may be necessary to expose attachment proteins on the virion surface before attachment to, and infection of, midgut cells can occur. This model may suggest answers to questions regarding the molecular basis for midgut infection barriers and species susceptibility to arbovirus infection in nature.

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.

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.

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.

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.

Structure and Function of the Immune System

The major function of the immune system is to protect the host from environmental agents such as microbes or chemicals, thereby preserving the integrity of the body. This is done by the recognition of self and response to non-self. The immune response has been artificially divided into innate immunity (resistance) and specific immunity. Specific immunity is further divided into humoral immunity, the one involved with antibody, and cellular immunity, which is orchestrated by T cells. It is essential to understand that although these divisions have helped in understanding and analyzing the immune response, the system functions as a single unit rather than as a separate entity. In this paper, a simplified analysis of specific immunity will be given. However, the importance of nonspecific immunity, especially as it pertains to its role in preventing exposure of environmental substances, should not be forgotten.

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 β1 or β4 integrin subunit. Substance P or inhibitors of the NK1 or NK2 tachykinin receptors also had no inhibitory effects on HSV-1 plaque formation.

Antigenic topography of bluetongue virus 17

Neutralizing monoclonal antibodies (mAbs) have been produced and used to map the topographical relationship of the surface antigenic determinants of bluetongue virus (BTV) 17 that mediate neutralization. Eight monoclonal antibodies, at least five of which were directed to the major outer coat protein of BTV 17, P2, were studied in neutralization assays using variant BTV 17 and in competition binding experiments. Five different epitopes were identified that are involved in neutralization of viral infectivity. Three of the five epitopes are clearly associated with P2, while the location of the other two epitopes is not known. The potential association of these two epitopes with one or both outer coat proteins of BTV is discussed.