A study regarding bovine enterovirus type 1 infection in domestic animals and humans: An evaluation from the zoonotic aspect.

Bovine enteroviruses (BEV) are members of Enterovirus genus of the family Picornaviridae. BEV1 has a broad host spectrum, including humans. The virus usually causes subclinical infection, but fatal/severe cases have also been reported in different animal species. There is quite limited data regarding BEV1 in humans. The purpose of this study is to investigate human infection and to identify possible risk factors for viral exposure. For this purpose, blood serum samples (n=1,526) were collected from a city center and nearby villagers simultaneously from humans and farm animals in Elazig province in Eastern Anatolia. As a result of serum neutralisation test, BEV1 specific antibody presence detected in cattle was 85.3% (163/191), 73.5% in donkeys (64/87), 71.8% in goats (115/160), 46.5% in sheep (93/200), 43.9% in horses (40/91), 41.3% in dogs (19/46) and 33% in humans (248/751). Although a high contamination potential was mentioned for people living in rural areas, it was determined that infection rates in rural areas (31.6%) and urban centers (32.2%) were very close. There was no difference according to sex. Viral exposure is higher in the 40 to 70 age range. In addition, the serological evidence of the infection in donkeys was identified for the first time with this study.

Identification of three linear B cell epitopes using monoclonal antibodies against bovine enterovirus VP2 protein.

Bovine enterovirus (BEV) VP2 protein is a structural protein that plays an important role in inducing protective immunity in the host. The function of VP2 has been characterized, but there is little information on its B cell epitopes. Three monoclonal antibodies (mAbs) directed against BEV VP2 were generated and characterized from mice immunized with the recombinant VP2 protein. Three minimal linear epitopes 152FQEAFWLEDG161, 168LIYPHQ173, and 46DATSVD51 reactive to the three mAbs were identified using western blotting analysis. Three-dimensional model of the BEV-E virion and the VP2 monomer showed that epitope 152FQEAFWLEDG161 is exposed on surface of the virion and epitopes 46DATSVD51 and 168LIYPHQ173 are located inside the virion. Alignment of the amino acid sequences corresponding to the regions containing the three minimal linear epitopes in the VP2 proteins and their cross-reactivity with the three mAbs showed that epitope 168LIYPHQ173 is completely conserved in all BEV strains. Epitope 46DATSVD51 is highly conserved among BEV-E strains and partly conserved among BEV-F strains. However, epitope 152FQEAFWLEDG161 is not conserved among BEV-F strains. Using the mAbs of 3H4 and 1E10, we found that VP2 localized in the cytoplasm during viral replication and could be used to monitor the viral antigen in infected tissues using immunohistochemistry. A preliminary 3H4-epitope-based indirect ELISA allowed us to detect anti-BEV-strain-HY12 antibodies in mice. This study indicates that the three mAbs could be useful tools for investigating the structure and function of the viral VP2 protein and the development of serological diagnostic techniques for BEV infection.

Rescue and characterization of a recombinant HY12 bovine enterovirus carrying a foreign HA epitope in the 3A nonstructural protein.

Full-length infectious cDNA clones for recombinant HY12 bovine enteroviruses designated as rHY12-3A-2-HA, rHY12-3A-3-HA, and rHY12-3A-9-HA were constructed by the insertion of an epitope from influenza virus hemagglutinin (HA) at the N-terminus of the HY12-encoded 3A protein at amino acid positions 2, 3, and 9. The recombinant HY12 viruses expressing the HA epitope were rescued and characterized using immunoperoxidase monolayer assay, western blotting, and electron microscopy. The three rescued recombinant marker viruses showed similar characteristics, such as TCID50 titer, plaque size, and growth properties, to those of parental rHY12 virus. Comparative analysis of the nucleotide sequences demonstrated the three recombinant marker viruses remained stable for 15 passages with no genetic changes. The recombinant viruses remained viable in various permissive cell lines, including BHK-21, Vero, and PK15 cells, suggesting that the insertion of the HA epitope tag had no effect on virus infectivity. Mice infected with the recombinant marker viruses and the parental virus produced anti-HY12-virus antibodies, while the recombinant marker viruses also produced anti-HA-epitope-tag antibodies. Taken together, these results demonstrate that HY12 viruses containing genetic markers may be useful tools for future investigations of the mechanisms of viral pathogenesis and virus replication, as well as for vaccine development.

VP1 B-C and D-E loops of bovine enterovirus cluster B can effectively display foot-and-mouth disease virus type O-conserved neutralizing epitope.

On the basis of generation of an infectious cDNA clone for the BHM26 strain of bovine enterovirus cluster B (BEV-B), 22 sites on different loops of the BHM26 capsid were selected according to an alignment of its sequence with the structural motifs of BEV-A strain VG-5-27 for insertion of the foot-and-mouth disease virus (FMDV) type O-conserved neutralizing epitope 8E8. Two recombinant viruses, rBEV-A1 and rBEV-DE, in which the FMDV epitope was inserted into the VP1 B-C or D-E loops, were rescued by transfection of BHK-21 cells with the in vitro-transcribed RNA of the recombinant BHM26 genome-length cDNA constructs. The two epitope-inserted viruses were genetically stable and exhibited growth properties similar to those of their parental virus in BHK-21 and IBRS-2 cells, which are susceptible to both BEV and FMDV. However, the two recombinant BEVs (rBEVs) had a significantly lower growth titre than those of the parental virus BHM26 in MDBK and Marc145 cells, which are susceptible to BEV but not to FMDV. These results indicated that insertion of the FMDV epitope into the VP1 B-C or D-E loops of the BEV particle altered the replication properties of BEV. In addition, the two rBEVs were sensitive to neutralization by the FMDV type O-specific mAb 8E8, and anti-FMDV IgG antibodies were induced in mice by intramuscular inoculation with the rBEV-A1 and rBEV-DE viruses. Our results demonstrate that the VP1 B-C and D-E loops of the BEV-B particle can effectively display a foreign epitope, making this an attractive approach for the design of BEV-vectored and epitope-based vaccines.

Pathogenesis of a bovine enterovirus-1 isolate in experimentally infected calves.

The pathogenesis and virulence of Bovine enterovirus-1 (BEV-1) in cattle is largely unknown. Reports concerning its virulence suggest that there might be an association between BEV-1 infections and a range of diseases in cattle that vary from respiratory to enteric to reproductive disease and infertility. In the current study, the pathogenesis associated with acute infection of BEV-1 in calves experimentally inoculated with the Oklahoma isolate of BEV-1 was described. Although interpretation of the study was limited by lack of an effective control group, results suggest that an association between inoculation of BEV-1, virus localization, and the potential development of lesions in the brain and heart probably exists. In the experiment, BEV-1 virus localized to the terminal ileum, ileocecal and cecocolonic junctions, spiral colon, and ileocecal lymph nodes; BEV-1 virus was detected in the cytoplasm of enterocytes, lamina propria macrophages, endothelium, neurons of the submucosal and myenteric plexi, and lymphocytes of the submucosal lymphoid tissue. Although no clinical signs were noted following acute infection, BEV-1 was localized in the cerebellar white matter of a calf with encephalitis and in the heart of another calf with coronary arteritis. The current study suggests that the BEV-1 isolate is infectious to young calves and that BEV-1 potentially can have a similar pathogenesis to that observed in natural or experimental enterovirus infections in other species.

Serological survey of bovine enterovirus type 1 in different mammalian species in Turkey.

The bovine enterovirus type 1 (BEV-1) infection has a wide range of host spectrum including humans. In this study, seroprevalence of BEV-1 was investigated in eight mammalian species. Blood serum samples were collected from 244 humans, 1520 cattle, 272 horse, 126 dog, 281 sheep, 477 goat, 18 camel (Camelus dromedarius) and 82 gazelle (Gazella subgutturosa subgutturosa) in different regions of Turkey. Microneutralization tests showed that gazelle and camel did not have any seropositivities, but seropositivities were detected in humans (30.3%), cattle (64.8%), horse (12.8%), dog (3.2%), sheep (32.8%) and goat (27.6%).