However, the studies differed in the severity of IAV infection and timing between viruses. mice given a nonlethal dose of MHV-1. RV-primed mice had reduced pulmonary inflammation and hemorrhage and influx of leukocytes, especially neutrophils, in Ropinirole HCl the airways upon MHV-1 infection. Although MHV-1 replication was reduced in RV-primed mice, RV did not inhibit MHV-1 replication in coinfected lung epithelial cells test with Holm-Sidak multiple-comparison correction. Viral titers and qPCR data were compared between groups using Students tests without correction for multiple comparisons. Statistical analysis of transcriptome data is Ropinirole HCl described above. Results Inoculation With RV Reduces Morbidity and Prevents Mortality of a Lethal MHV-1 Infection Based on our previous finding that BALB/c mice infected with 2×103 PFU of MHV-1 experienced 20% mortality (14), we inoculated mice with 2×105 PFU of MHV-1. This dose of MHV-1 resulted in 100% lethality ( Figure?1A ). In comparison to mice that received a mock inoculation two days before MHV-1 (mock/MHV), those that received RV (RV/MHV) were completely protected from mortality ( Figure ?1A ). RV/MHV infected mice also had less severe morbidity, as?determined by weight loss and clinical scores, compared to mock/MHV infected mice ( Figures?1B, C ). Although RV/MHV infected mice experienced significant weight loss, the rate of loss was lower than mock/MHV infected mice and they began regaining their body weight by day 7 after MHV-1 infection. Clinical signs of disease were delayed by two days and were much less severe in RV/MHV compared to mock/MHV infected mice. Clinical signs in mock/MHV infected mice included severely ruffled fur and hunched posture with mild to moderate lethargy and labored or shallow breathing. In contrast, clinical signs in RV/MHV infected mice were limited to mildly ruffled fur and hunched posture with occasional shallow breathing. Mock/MHV infected mice were Rabbit Polyclonal to ATP7B humanely euthanized or succumbed to infection on days 4-7, while all RV/MHV infected mice survived through the end of the study (day 14). Open in a separate window Figure?1 Priming with RV reduces morbidity and prevents mortality upon MHV-1 infection. Mice (n=7 per group) were inoculated intranasally with RV (7.6×106 TCID50) or saline (mock) on day -2, and MHV-1 (2×105 PFU) on day 0. Mice were monitored daily for (A) mortality ((8). We tested whether RV would inhibit MHV-1 infection in a Ropinirole HCl murine lung epithelial cell line, LA4. LA4 cells were inoculated with MHV-1 and RV concurrently or sequentially with RV 6? h prior to MHV-1 ( Figure?6 ). In contrast to our findings ( Figure?2 ), RV did not inhibit replication of MHV-1 either during concurrent or sequential coinfection ( Figures?6A, B ). In order to determine whether RV Ropinirole HCl and MHV-1 were infecting Ropinirole HCl the same cells within a coinfected culture, we performed IFA for viral antigens 18?h after concurrent coinfection. As we have previously shown, MHV-1 formed syncytia among infected cells, while cells infected with RV alone were dramatically condensed (21). Several cells contained antigens from both viruses ( Figure?6C , arrows), indicating that neither virus inhibited super-infection of the cell by the other virus. Open in a separate window Figure?6 RV does not inhibit MHV-1 replication in a coinfected epithelial cell line. LA4 cells were inoculated with (A) RV and MHV-1 concurrently or (B) RV 6 hours before MHV-1. Supernatant media from triplicate samples per time point were titrated for MHV-1 by TCID50 assay using 17Cl.1 cells. (C) LA4 cells were inoculated with RV and MHV-1 concurrently, and viral antigens were labelled by IFA 18 hours later. Antibodies against RV were detected with Alexa488 (green) and MHV-1 with Alexa555 (red) and nuclei were labelled with DAPI (blue). The panels show RV (green), MHV-1 (red), and overlay of both images. White arrows show examples of coinfected cells containing both RV and MHV-1 antigens. RV Dominates the Transcriptional Response of Mouse Lung Epithelial Cells Over That of MHV-1 To understand how coinfection by MHV-1 and RV affects gene expression in epithelial cells, LA4 cells were inoculated with MHV-1 alone, RV alone, or coinfected with both viruses (concurrently and sequentially) and total gene expression was analyzed using microarrays. Genes were more dramatically up- or down-regulated by RV infection at both 12 and 24?h (RV12; RV24) time points compared to MHV-1 (MHV12; MHV24; Figure?7 ). Cells coinfected with both viruses for 12?h (MHV12+RV12) had a similar gene expression profile to those infected by RV for 12?h (RV12). The difference in gene expression levels vs. mock was increased in cells infected with MHV-1 for 12?h.