Examining innate responses to Flaviviridae infection: how myeloid cells respond to and are modulated by bovine viral diarrhea virus in vivo and in vitro.

Disease associated with BVDV infection can be devastating ranging to millions of dollars in loss to the cattle industry. As well, infection with BVDV leads to an immune-suppressed host though an unknown mechanism(s). Although there are vaccines available, they are not broadly protective and ineffective in PI cattle. The myeloid lineage cells such as monocytes and MΦ are important in recognizing BVDV as well as initiating an adaptive immune response. There is little information on the effects of BVDV in vivo on the functionality of these cells and a better understanding can lead to potential treatment. Investigating the effects on cytokine production, expression and response of TLRs and functional aspects of phagocytosis are critical. The specific aims in this dissertation studies utilize neonatal calves for in vivo study and 1-year-old cattle for cell donors during in vitro investigation. BVDV-2 strain 1373 was utilized as it is a field strain, which induces severe acute disease and is a highly virulent. BVDV-2 strains RS886 and 28508 are subclinical acute disease inducing strains, which are lower in virulence and typical of field isolates from PI cattle or subclinical infections. BVDV-2 strain 296c is a cytopathic strain whereas BVDV-2 strain 296nc is an isogenic non-cytopathic strain. These isogenic strains were chosen as the only differences between the strains are the NS2/3 coding regions. BVDV-2 strains 1373 and RS886 were utilized for in vivo experimentation whereas BVDV-2 strains 1373, 28508, 296c and 296nc were used for in vitro experiments. Both in vivo and in vitro models were used to characterize viral effects on cytokine expression and secretion, TLR responsiveness, signaling events, and phagocytosis of myeloid cells after exposure to virus

Bovine Viral Diarrhea Virus Type 2 Impairs Macrophage Responsiveness to Toll-Like Receptor Ligation with the Exception of Toll-Like Receptor 7

<div><p><i>Bovine viral diarrhea virus</i> (BVDV) is a member of the <i>Flaviviridae</i> family. BVDV isolates are classified into two biotypes based on the development of cytopathic (cp) or non-cytopathic (ncp) effects in epithelial cell culture. BVDV isolates are further separated into species, BVDV1 and 2, based on genetic differences. Symptoms of BVDV infection range from subclinical to severe, depending on strain virulence, and may involve multiple organ systems and induction of a generalized immunosuppression. During BVDV-induced immune suppression, macrophages, critical to innate immunity, may have altered pathogen recognition receptor (PRR) signaling, including signaling through toll-like receptors (TLRs). Comparison of BVDV 2 strains with different biotypes and virulence levels is valuable to determining if there are differences in host macrophage cellular responses between viral phenotypes. The current study demonstrates that cytopathic (cp), noncytopathic (ncp), high (hv) or low virulence (lv) BVDV2 infection of bovine monocyte-derived macrophages (MDMΦ) result in differential expression of pro-inflammatory cytokines compared to uninfected MDMΦ. A hallmark of cp BVDV2 infection is IL-6 production. In response to TLR2 or 4 ligation, as might be observed during secondary bacterial infection, cytokine secretion was markedly decreased in BVDV2-infected MDMΦ, compared to non-infected MDMΦ. Macrophages were hyporesponsive to viral TLR3 or TLR8 ligation. However, TLR7 stimulation of BVDV2-infected MDMΦ induced cytokine secretion, unlike results observed for other TLRs. Together, these data suggest that BVDV2 infection modulated mRNA responses and induced a suppression of proinflammatory cytokine protein responses to TLR ligation in MDMΦ with the exception of TLR7 ligation. It is likely that there are distinct differences in TLR pathways modulated following BVDV2 infection, which have implications for macrophage responses to secondary infections.</p></div

Expression of proinflammatory cytokine gene transcription in MDMΦs inoculated with high and low virulence BVDV2 strains.

<p>MDMΦs were differentiated in 96 well plates for 7 days and inoculated with BVDV2 strains in duplicate at an MOI of 1 with RNA harvested at 2, 6, 18, and 24 h after inoculation. Cytokine mRNA was analyzed by qPCR using ribosomal protein s9 (RPS9) as an endogenous control with fold change expressed relative to uninfected control MDMΦs harvested at the corresponding time points. <i>il1β</i> (A), <i>tnfα</i> (B), <i>il6</i> (C), <i>il8</i> (D), <i>il12p40</i> (E), <i>il10</i> (F) were measured using primer sets specific for bovine genes using SYRB Green chemistry. Bars represent the mean value ± SEM from four different experiments from 9 total donor cattle. *** P < 0.001.</p

Proinflammatory cytokine secretion of BVDV2 inoculated or LPS stimulated MDMΦs 24 h after treatment.

<p>MDMΦs were differentiated in 96 well plates for 7 days and inoculated with BVDV2 strains in duplicate at an MOI of 1 or 2 μg/mL LPS with cell supernatants harvested at 24 h after treatment. Cytokine protein was analyzed by Searchlight Array using analytes specific for bovine IL-1β (A), IL-6 (B) and TNFα (C) with 50 μL of cell supernatant analyzed in duplicate. Cytokines were quantified by generation of standard curves against recombinant bovine cytokines provided by the manufacturer of the Searchlight platform. Bars represent the mean value ± SEM from four different experiments from 9 total donor cattle. ** P < 0.001.</p

Primer sequence for bovine targets and control genes.

<p>Primer sequence for bovine targets and control genes.</p

TNFα protein secretion from BVDV2 infected monocyte derived macrophages after stimulation with bacterial TLR agonists.

<p>MDMΦs were differentiated in 96 well plates for 7 days and inoculated with BVDV2 strains with an MOI of 1 for 48 h prior to stimulation with Pam3Cys [5 μg/mL], <i>M</i>. <i>haemolytica</i> LPS [10 μg/mL], or <i>E</i>. <i>coli</i> (055:B5) LPS [1 μg/mL]. Cell supernatants were analyzed for TNFα protein concentration 24 h after TLR stimulation and measured by Searchlight Array platform. Data from infection with cytopathic or noncytopathic strains are in the top panel (A) and from high or low virulence strains indicated in the lower panel (B). Bars represent the mean value ± SEM from four different experiments from 9 total donor cattle. ** P < 0.001 compared to uninfected, TLR stimulated cells.</p

TNFα protein secretion from BVDV2 infected MDMΦ after stimulation with viral TLR agonists.

<p>MDMΦs were differentiated in 96 well plates for 7 days and inoculated with BVDV2 strains with an MOI of 1 for 48 h prior to stimulation with Poly I:C [50 μg/mL], Imiquimod [10 μg/mL], ssRNA40 LyoVec [10 μg/mL]. Cell supernatants were analyzed for TNFα protein concentration 24 h after TLR stimulation and measured by Searchlight Array platform. Incubation with cytopathic and noncytopathic strains are in the top panel (A) or high and low virulence strains indicated in the lower panel (B). Bars represent the mean value ± SEM from four different experiments from 9 total donor cattle. ** P < 0.001 compared to uninfected, TLR stimulated cells.</p

Cattle intestinal microbiota shifts following Escherichia coli O157:H7 vaccination and colonization

Vaccination-induced Escherichia coli O157:H7-specific immune responses have been shown to reduce E. coli O157:H7 shedding in cattle. Although E. coli O157:H7 colonization is correlated with perturbations in intestinal microbial diversity, it is not yet known whether vaccination against E. coli O157:H7 could cause shifts in bovine intestinal microbiota. To understand the impact of E. coli O157:H7 vaccination and colonization on intestinal microbial diversity, cattle were vaccinated with two doses of different E. coli O157:H7 vaccine formulations. Six weeks post-vaccination, the two vaccinated groups (Vx-Ch) and one non-vaccinated group (NonVx-Ch) were orally challenged with E. coli O157:H7. Another group was neither vaccinated nor challenged (NonVx-NonCh). Fecal microbiota analysis over a 30-day period indicated a significant (FDR corrected, p 0.05) was not associated with vaccination but the relative abundance of Proteobacteria was significantly lower (p < 0.05) in vaccinated calves after E. coli O157:H7 challenge. Similarly, Vx-Ch calves had higher relative abundance of Paeniclostridium spp. and Christenellaceae R7 group while Campylobacter spp., and Sutterella spp. were more abundant in NonVx-Ch group post-E. coli O157:H7 challenge. Only Vx-Ch calves had significantly higher (p < 0.001) E. coli O157:H7-specific serum IgG but no detectable E. coli O157:H7-specific IgA. However, E. coli O157:H7-specific IL-10-producing T cells were detected in vaccinated animals prior to challenge, but IFN-γ-producing T cells were not detected. Neither E. coli O157:H7-specific IgG nor IgA were detected in blood or feces, respectively, of NonVx-Ch and NonVx-NonCh groups prior to or post vaccinations. Both Vx-Ch and NonVx-Ch animals shed detectable levels of challenge strain during the course of the study. Despite the lack of protection with the vaccine formulations there were detectable shifts in the microbiota of vaccinated animals before and after challenge with E. coli O157:H7

Vectorborne Transmission of Leishmania infantum from Hounds, United States

Leishmaniasis is a zoonotic disease caused by predominantly vectorborne Leishmania spp. In the United States, canine visceral leishmaniasis is common among hounds, and L. infantum vertical transmission among hounds has been confirmed. We found that L. infantum from hounds remains infective in sandflies, underscoring the risk for human exposure by vectorborne transmission