Barbara A. Israel

Human adenovirus-36 is associated with increased body weight and paradoxical reduction of serum lipids

BACKGROUND:Human adenovirus-36 (Ad-36) increases adiposity and paradoxically lowers serum cholesterol and triglycerides in chickens, mice, and non-human primates. The role of Ad-36 in human obesity is unknown.OBJECTIVES:To determine the prevalence of Ad-36 antibodies in obese and nonobese humans. To evaluate the association of Ad-36 antibodies with body mass index (BMI) and serum lipids.DESIGN:Cohort study. Volunteers from obesity treatment programs, communities, and a research study.SUBJECTS:Obese and nonobese volunteers at the University of Wisconsin, Madison, WI, and the Bowen Center, Naples, Florida. Obese and thin volunteer research subjects and 89 twin pairs at Columbia University, New York.INTERVENTIONS:Study 1: 502 subjects; serum neutralization assay for antibodies to Ad-2, Ad-31, Ad-36, and Ad-37; serum cholesterol and triglycerides assays. Study 2: BMI and %body fat in 28 twin pairs discordant for Ad-36 antibodies.MAIN OUTCOME MEASURES:Presence of antibodies to adenoviruses, BMI, serum cholesterol and triglycerides levels.RESULTS:Significant (P<0.001) association of obesity and positive Ad-36 antibody status, independent of age, sex, and collection site. Ad-36 antibodies in 30% of obese, 11% of nonobese. Lower serum cholesterol and triglycerides (P<0.003) in Ad-36 antibody-positive vs -negative subjects. Twin pairs: antibody-positive twins had higher BMIs (24.5±5.2 vs 23.1±4.5 kg/m2, P<0.03) and %body fat (29.6±9.5% vs 27.5±9.9%, P<0.04). No association of Ad-2, Ad-31, or Ad-37 antibodies with BMI or serum lipids.CONCLUSIONS:Ad-36 is associated with increased body weight and lower serum lipids in humans. Prospective studies are indicated to determine if Ad-36 plays a role in the etiology of human obesity.

Increased adiposity in animals due to a human virus

BACKGROUND: Four animal models of virus-induced obesity including adiposity induced by an avian adenovirus have been described previously. This is the first report of adiposity induced in animals by a human virus.OBJECTIVE: We investigated the adiposity promoting effect of a human adenovirus (Ad-36) in two different animal models.DESIGN: Due to the novel nature of the findings we replicated the experiments using a chicken model three times and a mammal model once. In four separate experiments, chickens and mice were inoculated with human adenovirus Ad-36. Weight matched groups inoculated with tissue culture media were used as non-infected controls in each experiment. Ad-36 inoculated and uninfected control groups were housed in separate rooms under biosafety level 2 or better containment. The first experiment included an additional weight matched group of chickens that was inoculated with CELO (chick embryo lethal orphan virus), an avian adenovirus. Food intakes and body weights were measured weekly. At the time of sacrifice blood was drawn and visceral fat was carefully separated and weighed. Total body fat was determined by chemical extraction of carcass fat.RESULTS: Animals inoculated with Ad-36 developed a syndrome of increased adipose tissue and paradoxically low levels of serum cholesterol and triglycerides. This syndrome was not seen in chickens inoculated with CELO virus. Sections of the brain and hypothalamus of Ad-36 inoculated animals did not show any overt histopathological changes. Ad-36 DNA could be detected in adipose tissue, but not skeletal muscles of randomly selected animals for as long as 16 weeks after Ad-36 inoculation.CONCLUSIONS: Data from these animal models suggest that the role of viral disease in the etiology of human obesity must be considered.

Transmissibility of adenovirus-induced adiposity in a chicken model.

BACKGROUND: We previously reported that human adenovirus Ad-36 induces adiposity and paradoxically lower levels of serum cholesterol (CHOL) and triglycerides (TG) in animals.OBJECTIVE: To evaluate the transmissibility of Ad-36 and Ad-36 induced adiposity using a chicken model.DESIGN: Experiment 1—four chickens were housed (two per cage) and one from each cage was inoculated with Ad-36. Duration of presence of Ad-36 DNA in the blood of all chickens was monitored. Experiment 2—two groups of chickens were intranasally inoculated with Ad-36 (infected donors, I-D) or media (control donors, C-D). Blood drawn 36 h later from I-D and C-D groups was inoculated into wing veins of recipient chickens (infected receivers, I-R, and control receivers, C-R, respectively). On sacrifice, 5 weeks post-inoculation, blood was drawn, body weight noted and visceral fat was separated and weighed.RESULTS: Experiment 1—Ad-36 DNA appeared in the blood of the inoculated chickens and that of uninoculated chickens (cage mates) within 12 h of inoculation and the viral DNA persisted up to 25 days in the blood. Experiment 2—compared with C-D, visceral and total body fat were significantly greater and CHOL significantly lower for the I-D and I-R. TG were significantly lower for the I-D. Ad-36 was isolated from 12 out of 16 blood samples of the I-D that were used for inoculating I-R chickens. Ad-36 DNA was present in the blood and the adipose tissue of the I-D and I-R but not in the skeletal muscles of animals selected randomly for testing.CONCLUSION: As seen in experiment 1, Ad-36 infection can be transmitted horizontally from an infected chicken to another chicken sharing the cage. Additionally, experiment 2 demonstrated blood-borne transmission of Ad-36-induced adiposity in chickens. Transmissibility of Ad-36-induced adiposity in chicken model raises serious concerns about such a possibility in humans that needs further investigation.

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.

Induction of a mucosal barrier to bovine herpesvirus 1 replication in cattle

Current vaccines for human and animal herpesviruses engender an immunity that may ameliorate disease but generally fails to prevent infection, latency, reactivation from latency, or spread through a population. By administering intranasally to cattle bovine herpesvirus type 1 virion envelope proteins combined with the potent mucosal immune system adjuvant, cholera toxin B subunit, we engendered a local antibody response that acted as a barrier to infection of mucosal epithelial cells and thereby prevented viral replication, consequently precluding disease, latency, and spread.

Monoclonal antibody analysis of bovine herpesvirus-1 glycoprotein antigenic areas relevant to natural infection

Neutralizing antigenic areas on the glycoproteins of bovine herpesvirus-1 (BHV-1) were identified by reciprocal competition radioimmunoassays using monoclonal antibodies. Three interrelated and two independent antigenic areas were identified on the 77-kDa (K) gIV envelope glycoprotein. Antigenic analysis of this protein has not been previously described. Four interrelated and one independent antigenic areas were found on the 97K gIII envelope glycoprotein. A third group of monoclonal antibodies reacting in Western blot with the 74K subunit of gI, a 130K disulfide-linked 74K/55K heterodimer, revealed four interrelated antigenic areas. All of the antigenic areas on all three glycoproteins were reactive with neutralizing monoclonal antibodies and all were targets for antibody-complement lysis. However, antibodies against gIV were the most efficient at neutralizing the virus and rendering infected cells susceptible to antibody-complement lysis. Convalescent sera from experimentally infected calves were used in a competitive radioimmunoassay to confirm that each antigenic area on the gI, gIII, or gIV glycoproteins was a target for bovine antibodies during primary infection with BHV-1.

Effects of formalin inactivation on bovine herpes virus-1 glycoproteins and antibody response elicited by formalin-inactivated vaccines in rabbits

The possibility of using a radioimmune assay (RIA) to measure the retention of antigenic determinants on gI, gIII, and gIV, the three major bovine herpes virus 1 (BHV-1) glycoproteins, during the formalin inactivation and aluminium hydroxide adsorption commonly used in the manufacture of killed virus vaccines was investigated. Monoclonal antibodies were used to measure thirteen previously identified glycoprotein epitopes in experimental vaccines containing live BHV-1 or formalin-killed BHV-1 and a commercial formalin-inactivated BHV-1 vaccine. All four epitopes on gI lost 80-97% of their ability to bind monoclonal antibodies. Two of five epitopes on gIII were preserved but the other three epitopes on gIII and five on gIV were reduced as much as 88%. To show that the measured loss of epitopes was biologically relevant, the vaccines were administered to rabbits and the resulting antibody responses against the same epitopes were evaluated by competition RIA. Little correlation was found between the measured antigenic site destruction and the rabbit response against those antigenic sites, suggesting that monoclonal antibody reactivity of a formalin-inactivated vaccine could not provide a meaningful measure of antigenic activity.

Epitope specificity and protective efficacy of the bovine immune response to bovine herpesvirus-1 glycoprotein vaccines

Bovine herpesvirus-1 (BHV-1) envelope glycoproteins gI, gIII and gIV were individually purified on monoclonal antibody affinity columns and injected intradermally into BHV-1 seronegative calves. The calves developed serum neutralizing antibodies that monospecifically precipitated the immunizing glycoprotein from a preparation of 125I-labelled BHV-1 envelope proteins. A competitive radioimmunoassay using the bovine antisera demonstrated that known functional epitopes had been retained in the glycoprotein vaccines. Calves immunized with the gI, gIII or gIV glycoproteins were not protected from intranasal challenge with BHV-1 and had levels and duration of viral shedding in their nasal secretions similar to those of non-immunized control calves.

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.