Identification and Expression of Immunogenic Proteins of a Disease-Associated Marine Turtle Herpesvirus

Abstract

Herpesviruses are associated with several diseases of marine turtles, including lung-eye-trachea disease (LETD) and fibropapillomatosis. Two approaches were used to identify immunodominant antigens of LETV, the LETD-associated herpesvirus. The first approach targeted glycoprotein B, which is known to be immunogenic and neutralizing in other species. The second strategy identified LETV proteins recognized on Western blots by antibodies in immune green turtle plasma. A 38-kDa protein was resolved by two-dimensional gel electrophoresis, sequenced, and identified as a scaffolding protein encoded by the overlapping open reading frames of UL26 and UL26.5. Glycoprotein B and the scaffolding protein were cloned and expressed in Escherichia coli. The expressed proteins were recognized on Western blots by antibodies in immune green turtle plasma. Phylogenetic studies based on UL26, DNA polymerase, and glycoprotein B revealed that LETV clusters with the alphaherpesviruses.Herpesviruses are associated with several diseases of marine turtles, including lung-eye-trachea disease (LETD) and fibropapillomatosis (FP). Recent efforts to understand the role of these herpesviruses and their impact on threatened and endangered marine turtle populations have focused on the development of serological assays for seroepidemiological studies. An LETD-associated herpesvirus (LETV)-specific enzyme-linked immunosorbent assay (ELISA) has been developed by using LETV-infected cell lysates as the antigen for detection of antibodies. This assay demonstrated that 21.6% of juvenile green turtles along the east coast of Florida were exposed to LETV or an LETV-like herpesvirus (3). In addition, 12 out of 13 nesting green and loggerhead turtles were shown to be seropositive for antibodies to LETV, suggesting that, as is the case with many human and mammalian herpesvirus infections, individuals are exposed at some point during their lifetimes. Clinical signs consistent with LETD have been observed in juvenile green turtles in the wild (8), and LETV has been found to be associated with LETD lesions in green turtles reared in captivity (11).LETV is a relatively low-titer and slow-growing virus. Generation of antigen for the LETV ELISA is labor intensive and subject to batch-to-batch variation. Expressed recombinant herpesvirus proteins could provide a source of antigen that would permit more extensive seroepidemiological studies of LETV infections. In this study, two strategies were used to select LETV proteins for expression. One protein was previously identified as a potential immunodominant LETV antigen (2, 3). This 38-kDa protein was recognized by many, but not all, individual wild turtles with antibodies to LETV and was targeted for cloning and expression. A second LETV protein, glycoprotein B (gB), was also selected for cloning and expression. gB has been identified as a major target of the antiherpesvirus immune response in many host species and includes virus-neutralizing activity (6,12,19). gB also has been extensively targeted for the developm...

The order Herpesvirales

“…In addition, several new species were classified at the level of subfamily or family, with assignment to genera awaiting further data. These include the reptilian species Chelonid herpesvirus 5 and Chelonid herpesvirus 6 in the subfamily Alphaherpesvirinae [3, 11, 13, 17, 26], Phocid herpesvirus 2 in the subfamily Gammaherpesvirinae [10, 12], and Iguanid herpesvirus 2 in the family Herpesviridae [13, 25]. …”

Genomic Variation of the Fibropapilloma-Associated Marine Turtle Herpesvirus across Seven Geographic Areas and Three Host Species

“…The lung-eye-trachea disease virus (LETV) sequences used for comparisons below are U L 26/U L 26.5 (AY124578), U L 27 (AY124557), and a partial sequence of U L 30 (pol) (AAM95779) (5).…”

Use of Baculovirus-Expressed Glycoprotein H in an Enzyme-Linked Immunosorbent Assay Developed To Assess Exposure to Chelonid Fibropapillomatosis-Associated Herpesvirus and Its Relationship to the Prevalence of Fibropapillomatosis in Sea Turtles

“…Some wild green turtles with FP also had antibody reactivity to LETHV-infected cells and culture lysates (7). One cross-reacting antigen, UL26 protease, has been identified (6).…”