Productive infection of field strains of avian coronavirus infectious bronchitis virus in chicken peripheral blood-derived monocyte

Yueting Zhang; Gary Whittaker

Peripheral blood-derived monocytes from chickens are infected by IBV. To investigate a role for myeloid cells in IBV dissemination and viral pathogenesis, we isolated peripheral blood-derived monocytes from chickens (chPBMCs) and confirmed that they were positive for the monocyte/macrophage marker KUL01 (Fig.1A) . Due to the lack of available reagents for avian species, we were unable to distinguish whether chPBMCs were matured or differentiated into macrophages or dendritic cells, or to determine the composition of unique cell populations. We found that chPBMCs are highly susceptible to the IBV field strains M41, Cal99, Conn46, and Iowa97 (Fig.1B) . However, the laboratory--adapted strain Beaudette, which is not pathogenic in chickens, did not infect chPBMCs. Infections were also repeated with macrophages isolated from bone marrow of chickens, with similar results (not shown). To examine whether the stage of maturation of the chPBMCs had an effect on IBV infection, we tested chPBMCs at different days post--seeding (Fig.1C) . The percentage of infected cells increased with time, reaching a maximum at day 5. Beyond day 3--4, we observed a large proportion of giant multinucleated cells, as observed previously (12) . In all further experiments, we utilized day 3--4 cultures due to difficulty in imaging and quantifying the giant cells. We next determined whether IBV infection of chPBMCs produces progeny viral particles. RT--PCR was performed to detect released particles. Starting at 8h, we detected viral RNA in the supernatant, reaching a maximum at 24h post-infection. To determine whether these progeny virions were infectious, we performed an EID50 assay. The peak viral titer was 10 8 EID50/mL at 12h post--infection. At later times, the titer decreased slightly even though RT--PCR indicated more virus particles were present (Fig.1D) . The reduced infectivity may be due to aggregation of viral particles between 12 and 24 h post infection. IBV induces rapid apoptosis in chPMBCs. One feature of chPBMC infection that we wished to examine was whether IBV can induce apoptosis. To do this, we used a FLICA assay that employs an inhibitor irreversibly binding to activated caspase3/7. At 6h post--infection, there was an increase in the fluorescence signal in IBVM41-infected cells, compared to mock--infected cells. Addition of UV--inactivated IBV--M41 also resulted in an increase in fluorescence signal, indicating that replication--defective IBV is also able to trigger apoptosis ( Fig.2A) . Cellular DNA from mock infected or infected chPBMCs was also collected after 24h post--infection and analyzed by gel electrophoresis. DNA from infected cells was fragmented and displayed an apoptosis-characteristic laddering, while no obvious laddering was observed for mock--infected cells (Fig.2B) . We show here that field strains of IBV can productively infect monocytes/macrophages and induce apoptosis, yet the laboratory--adapted strain Beaudette does not infect. Our data suggest that IBV infection of blood--derived myeloid cells might be an important component in the pathogenesis of IBV, and be critical for viral dissemination.