West Nile virus (WNV) neuroinvasive disease threatens the health and well-being of horses and humans worldwide. Disease in horses and humans is remarkably similar. The occurrence of WNV disease in these mammalian hosts has geographic overlap with shared macroscale and microscale drivers of risk. Importantly, intrahost virus dynamics, the evolution of the antibody response, and clinicopathology are similar. The goal of this review is to provide a comparison of WNV infection in humans and horses and to identify similarities that can be exploited to enhance surveillance methods for the early detection of WNV neuroinvasive disease.

We investigated the interaction between the insect-specific virus, Piura virus (PIUV), and the arbovirus Zika virus (ZIKV) in Aedes albopictus cells. We performed coinfection experiments in C6/36 cells. Piura virus (Cor 33 strain, Colombia) and ZIKV (PRVABC58 strain, Puerto Rico) were co-inoculated into C6/36 cells using two multiplicity of infection (MOI) combinations: 0.1 for both viruses and 1.0 for ZIKV, 0.1 for PIUV. Wells were infected in triplicate with either PIUV and ZIKV coinfection, ZIKV-only, or PIUV-only. Mock infected cells served as control wells. The cell suspension was collected daily 7 days post-infection. Zika virus load was titrated by TCID₅₀ on Vero 76 cells. The ZIKV-only infection and PIUV and ZIKV coinfection experiments were also quantified by RT-qPCR. We also investigated whether ZIKV interfered in the PIUV replication. PIUV suppressed the replication of ZIKV, resulting in a 10,000-fold reduction in ZIKV titers within 3 days post-infection. PIUV viral loads were not reduced in the presence of ZIKV. We conclude that, when concurrently infected, PIUV suppresses ZIKV in C6/36 cells while ZIKV does not interfere in PIUV replication.

Mycoplasma spp. are facultative pathogens that contribute to the pathogenesis of multiple bovine diseases, including the bovine respiratory disease complex, and have been shown to form biofilms. Biofilm formation is associated with increased antibiotic resistance in many organisms, but accurate determination of antimicrobial susceptibility in biofilms is challenging. In Mycoplasma spp., antimicrobial susceptibility is routinely determined using metabolic pH-dependent color change. However, biofilm formation can lead to reduced metabolism, making interpretation of metabolic readouts difficult. Therefore, we developed and optimized a new flow cytometry-based method for antimicrobial susceptibility testing in biofilm-forming Mycoplasma, termed the live/dead antimicrobial susceptibility test (LD-AST). The LD-AST measures the proportion of live bacteria upon exposure to antibiotics, works robustly with both planktonic and biofilm cultures, and enables the determination of the minimum bactericidal concentration (MBC) for a given antibiotic. We used two strains of Mycoplasma bovis (Donetta PG45 and Madison) and two clinical Mycoplasma bovoculi isolates (MVDL1 and MVDL2) to determine the impact of biofilm growth on antimicrobial susceptibility for gentamicin, enrofloxacin, or tetracycline. All Mycoplasma strains were susceptible to all antibiotics when cultured as planktonic cells, with MBCs in the expected range. However, three out of four strains (Donetta PG45, MVDL1, and MVDL2) were completely resistant to all three antibiotics when newly adhered biofilms were analyzed, whereas M. bovis Madison gave variable results. For mature biofilms that were cultured for 4–5 days before antibiotic exposure, results also were variable, with some strains showing an increased resistance with certain antibiotics and a decreased resistance with others. Overall, these results are consistent with earlier reports that biofilms can exhibit increased antimicrobial resistance.

We investigated the interaction between the insect-specific virus, Piura virus (PIUV) and the arbovirus Zika virus (ZIKV) in Aedes albopictus cells. We performed coinfection experiments in C6/36 cells. Piura virus (Cor 33 strain, Colombia) and ZIKV (PRVABC58 strain, Puerto Rico) were co-inoculated into C6/36 cells using two MOI combinations: 0.1 for both viruses and 1.0 for ZIKV, 0.1 for PIUV. Wells were infected in triplicate with either PIUV and ZIKV coinfection, ZIKV-only, or PIUV-only. Mock infected cells served as control wells. The cell suspension was collected daily through 7 days post-infection. Zika virus load was titrated by TCID50 on Vero 76 cells. The ZIKV-only infection and PIUV and ZIKV coinfection experiments were also quantified by RT-qPCR. We also investigated whether ZIKV interfered in the PIUV replication. PIUV suppressed the replication of ZIKV resulting in a 10,000-fold reduction in ZIKV titers within 3 days post-infection. PIUV viral loads were not reduced in the presence of ZIKV. We conclude that, when concurrently infected, PIUV suppresses ZIKV in C6/36 cells while ZIKV does not interfere in PIUV replication.