Antibody-dependent enhancement of virus infection is a process whereby virus-antibody complexes initiate infection of cells via Fc receptor-mediated endocytosis. We sought to investigate antibody-dependent enhancement of feline infectious peritonitis virus infection of primary feline peritoneal macrophages in vitro. Enhancement of infection was assessed, after indirect immunofluorescent-antibody labelling of infected cells, by determining the ratio between the number of cells infected in the presence and absence of virus-specific antibody. Infection enhancement was initially demonstrated by using heat-inactivated, virus-specific feline antiserum. Functional compatibility between murine immunoglobulin molecules and feline Fc receptors was demonstrated by using murine anti-sheep erythrocyte serum and an antibody-coated sheep erythrocyte phagocytosis assay. Thirty-seven murine monoclonal antibodies specific for the nucleocapsid, membrane, or spike proteins of feline infectious peritonitis virus or transmissible gastroenteritis virus were assayed for their ability to enhance the infectivity of feline infectious peritonitis virus. Infection enhancement was mediated by a subset of spike protein-specific monoclonal antibodies. A distinct correlation was seen between the ability of a monoclonal antibody to cause virus neutralization in a routine cell culture neutralization assay and its ability to mediate infection enhancement of macrophages. Infection enhancement was shown to be Fc receptor mediated by blockade of antibody-Fc receptor interaction using staphylococcal protein A. Our results are consistent with the hypothesis that antibody-dependent enhancement of feline infectious peritonitis virus infectivity is mediated by antibody directed against specific sites on the spike protein.

Fifty-four monoclonal antibodies (MAbs) to feline infectious peritonitis virus (FIPV) were characterized according to protein specificity, immunoglobulin subclass, virus neutralization, reactivity with different coronaviruses, and ability to induce antibody-dependent enhancement (ADE) of FIPV infection in vitro. The MAbs were found to be specific for one of three structural proteins of FIPV. A total of 47 MAbs were specific for the 205-kDa spike protein (S), 3 MAbs were specific for the 45-kDa nucleocapsid protein (N), and 4 MAbs were specific for the 26-to 28-kDa membrane protein (M). The S-specific MAbs showed various degrees of cross-reactivity with strains of FIPV, feline enteric coronavirus, canine coronavirus, and porcine transmissible gastroenteritis virus. Nineteen S-specific MAbs neutralized FIPV. A total of 15 of the neutralizing MAbs induced ADE, and ali but 1 were of the immunoglobulin G2a subclass. The remaining four neutralizing MAbs that did not induce ADE were of the immunoglobulin Gl subclass. Two S-specific MAbs induced ADE but were nonneutralizing. None of the Nor M-specific MAbs was neutralizing or induced ADE. On the basis of the reactivity patterns of the MAbs with FIPV and related coronaviruses, it was concluded that there is a minimum of five neutralizing sites on S. In most instances, neutralizing MAbs were able to induce ADE, demonstrating a direct relationship between neutralization and enhancement. The difference in immunoglobulin subclass between neutralizing MAbs that induced ADE and those that did not induce ADE suggests that there may be a restriction in the immunoglobulin subclasses capable of mediating ADE.

SUMMARYA panel of murine monoclonal antibodies (MAbs) against the two major glycoproteins of bovine viral diarrhoea virus (BDV) was produced and assayed by serum neutralization, radioimmunoprecipitation (RIP) and immunoblotting. Based on their viral polypeptide specificity and on their ability to neutralize viral infectivity, the MAbs in the panel were divided into three classes : Class 1 MAbs reacted with the 56K to 58K glycoprotein and neutralized the virus, class 2 MAbs recognized the 56K to 58K glycoprotein but were not neutralizing, and class 3 MAbs reacted with the 48K glycoprotein and did not neutralize the virus. These results identify the 56K to 58K protein as one of the envelope glycoproteins of BDV. Evidence was obtained indicating that it is responsible for the induction of neutralizing antibodies. No large uncleared precursors of the 56K to 58K protein could be identified unequivocally by RIP of infected cell extracts, suggesting that this polypeptide is proteolytically processed cotranslationally. A subset of MAbs that reacted with BDV isolates of the noncytopathic biotypes yielded similar results, indicating that these findings are applicable to both biotypes of BDV.

A panel of monoclonal antibodies that recognize the two major glycoproteins of bovine viral diarrhea virus (BDV) was used to evaluate the antigenic relationship between cytopathic (CP) and noncytopathic (NCP) viruses isolated from cattle dead or dying from fatal BDV infections. Various unrelated BDV isolates were initially screened by indirect immunofluorescence with monoclonal antibodies directed against the 56to 58. and 48-kilodalton glycoproteins of the virus. A wide spectrum of reactivity that was independent of biotype was found. Biological clones of the same isolate showed only minor variations from the parental isolate, as did isolates taken from different animals located on the same farm. A similar analysis was repeated with pairs of CP and NCP viruses isolated from 16 unrelated clinical cases of BDV infection resulting in fatal disease. The reactivity patterns within individual pairs of isolates taken from the same animals were in most instances very similar and in some cases indistinguishable from one another. The results demonstrate that antigenic similarity between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree of variation in reactivity patterns between unrelated BDV isolates, the close antigenic similarity of CP BDV to the homologous NCP BDV of a given pair strongly suggests that CP BDV arises by mutation from NCP BDV.

Seven calves between 1 week and 2 months of age were infected with a noncytopathic field isolate of bovine viral diarrhea virus (BDV) in order to evaluate the effect of BDV infection on the concentration of circulating platelets in the blood. All calves were determined to be free of BDV and neutralizing antibodies to BDV before infection. Platelet counts were performed on a daily basis over a 30-day period beginning at the time of infection. By 2 weeks postinfection, all calves showed a significant drop in the number of circulating platelets and a marked hyperplasia of megakaryocytes in the bone marrow. In three of the seven calves, thrombocytopenia was severe (less than or equal to 5,000/microliters) for 1 to 6 days. In two of these three animals, extensive petechial and ecchymotic hemorrhages were observed on all mucosal surfaces and on various internal organs during the period of severe thrombocytopenia. BDV was consistently isolated from the platelets during the early phases of the infection, and viral antigen was occasionally detected on platelets by a fluorescent-antibody assay. The results demonstrate that BDV infection is associated with decreases in platelet numbers and suggest that platelets may serve as carriers of circulating virus.

The S glycoprotein of feline infectious peritonitis virus (FIPV) has been shown to contain the antigenic sites responsible for eliciting both neutralization and antibody-dependent enhancement. To determine the region of S responsible, overlapping DNA fragments spanning the entire S gene were cloned and expressed as fusion proteins by in vitro transcription and translation. Fusion proteins containing relevant epitopes were identified by radioimmunoprecipitation with neutralizing and enhancing FIPV-specific monoclonal antibodies (MAbs). A region spanning residues 509 to 673 reacted with most MAbs tested. Translation in the presence of microsomal membranes did not enhance reactivity, suggesting that glycosylation is not essential for recognition by the MAbs. To localize the antigenic sites further, several MAb-resistant (mar) mutants of FIPV were cloned and sequenced. Amino acid residues that contribute to the neutralizing and enhancing epitopes were localized to two regions, designated A1 and A2, which show partial overlap with the homologous antigenic site A of transmissible gastroenteritis virus. Site A1 contains residues 568 and 591 and is homologous with part of subsite Aa of transmissible gastroenteritis virus. Site A2 contains residues 643, 649, and 656. Double mutations in sites A1 and A2 were found in mar mutants derived from neutralizing and enhancing MAbs 23F4.5 and 18A7.4, while a single mutation in site A2 was found in a mar mutant derived from MAb 24H5.4, which is neutralizing but not enhancing. The data suggest that site A2, which includes residues 643 to 656, is a dominant neutralizing site of FIPV and that sites A1 and A2 may act in concert to induce antibody-dependent enhancement.

We have previously demonstrated antibody-dependent enhancement of feline infectious peritonitis virus (FIPV) infection of macrophages using both virus-specific antisera and monoclonal antibodies (MAbs) to the spike (S) protein of FIPV. To increase our understanding of this phenomenon, six representative MAbs from a previously documented group of 12 enhancing MAbs were used to identify epitopes that mediate antibodydependent enhancement of FIPV infectivity. Analysis of the results of kinetics-based competitive ELISA (KcELISA) among these six enhancing MAbs grouped the epitopes into two clusters. Because transmissible gastroenteritis virus (TGEV) and FIPV are so closely related antigenically, we also conducted K-cELISA experiments between the FIPV MAbs and TGEV S protein-specific MAbs for which the epitopes had previously been mapped to specific sites on the TGEV S protein. Results of these assays indicated that the two FIPV epitope clusters are homologues of the previously defined TGEV S protein sites A and E/F. In addition, two TGEV S protein-specific MAbs also induced antibody-dependent enhancement of FIPV infection of macrophages. This functional cross-reactivity provides further support for the close antigenic relationship between FIPV and TGEV. Our results provide a preliminary localization of several enhancing epitopes within the amino acid sequence of the FIPV S protein.

Disseminated aspergillosis in dogs has been associated with Aspergillus terreus or A. deflectus infection. We report a case of disseminated A. versicolor infection presenting as diskospondylitis, osteomyelitis, and pyelonephritis. The diagnosis was made based on clinical, radiographic, and pathological findings. The etiologic agent was identified by fungal culture and internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing. This is the first description of canine aspergillosis caused by A. versicolor. CASE REPORTA 31-kg, 2.5-year-old male castrated German shepherd dog was examined at the Texas A&M University Veterinary Medicine Teaching Hospital because of nonambulatory paraparesis, weight loss, and hyporexia for 3 months. At admission, the dog had a body temperature of 104.6°F, heart rate of 160 beats/min, respiratory rate of 80 breaths/min, and blood pressure of 173/99 mm Hg. Neurological examination revealed a hyperreflexive patellar reflex bilaterally. Motor function was absent in the right pelvic limb and questionable in the left. The patient had marked generalized muscle atrophy. A lateral radiograph of the cranial thorax revealed lysis and shortening of the first four sternebrae (Fig. 1A). The second and third sternebrae were most severely affected and had irregular margins with loss of their end plates. Lateral radiographs of the thoracic vertebral column showed lysis and shortening of the 9th (T9) and 10th (T10) thoracic vertebrae with loss of the end plates and spondylosis deformans ventrally (Fig. 1B). Narrowing of the intervertebral space, end plate sclerosis, and ventral spondylosis derformans were also found between the seventh and eighth thoracic vertebrae. The clinical diagnoses were diskospondylitis involving T9-T10, osteomyelitis of the sternum and left humerus, and T3-L3 myelopathy resulting in nonambulatory paraparesis. Suspected causes included disseminated aspergillosis, blastomycosis, coccidioidomycosis, bacterial infection, and neoplasia. Due to the poor prognosis, the dog was subsequently euthanized and a complete necropsy was performed.The major skeletal changes found at postmortem examination included marked bony proliferation of the cranial end of the sternum, extending from the first to the fourth sternebrae, with loss of the joint space between the second and third sternebrae ( Fig. 2A). A soft gray area of necrotizing osteomyelitis was in the center of the collapsed and fused sternebrae. In the thoracic vertebral column, there was loss of the intervertebral disk at T9-T10 with lysis of the associated vertebral end plates (Fig. 2B). The latter changes resulted in joint instability, overriding of the vertebral bodies, and spinal cord compression that was exacerbated with ventroflexion of the vertebral column. In the kidneys, there was dark red to purple mottling of the cortical region with dozens of white to tan areas throughout the cortex and medulla (Fig. 2C). The renal crests were ulcerated, and the pelvises were dilated and contained a small amount of cloudy fluid with ...