Identification of Host Factors for Rift Valley Fever Phlebovirus.

Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID50 assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication.

Identification of host factors for Rift Valley Fever Phlebovirus.

Background: Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. Methodology: To identify the host factors or genes essential for RVFV replication, we conducted a CRISPR-Cas9 knock-out screen in human A549 cells. We then validated the putative genes using siRNA-mediated knockdowns and CRISPR-Cas9-mediated knockout studies, respectively. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers by plaque assay or TCID50 assay. Findings: We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knockdowns found that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of LRP1 in RVFV replication was previously described in detail. Knock-out A549 cell lines were generated and used to dissect the effect of WRD7 on RVFV and another bunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knock-out cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24h) when compared to LACV which was affected an earlier replication phase (12h). Conclusion: In summary, we have identified WDR7 as an essential host factor for the replication of two relevant bunyaviruses, RVFV and LACV. Future studies will investigate the mechanistic role by which WDR7 facilitates Phlebovirus replication.

Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation.

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: σ32 and σ70. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing σ70 (HU, P28-Omp14 and P28-Omp19) and σ32 (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions.

Comparison of Rifamycins for Efficacy Against Mycobacterium avium Complex and Resistance Emergence in the Hollow Fiber Model System.

Rifamycins are integral part of the combination regimen for treatment of pulmonary Mycobacterium avium-complex [MAC] infection, but different practitioners prefer different rifamycins. The objective of the study was to compare microbial kill and resistance emergence of rifamycins using principles of pharmacokinetics/pharmacodynamics. First, we identified rifamycin MICs in 20 MAC isolates from patients followed by concentration-response studies in test-tubes. Next, we examined efficacy and resistance suppression of three doses of each rifamycin in the hollow fiber system model of pulmonary MAC [HFS-MAC], mimicking human like concentration-time profile of the drugs. HFS-MAC units were repetitively sampled for total and drug-resistant MAC burden and for drug concentration measurements. Inhibitory sigmoid E max model, linear regression, and analysis of variance was used for data analysis. For rifabutin 90% of isolates had MIC ≤ 0.125 mg/L while for both rifampin and rifapentine this was ≤2.0 mg/L. There was no statistically significant difference (p > 0.05) in maximal kill and effective concentration mediating 50% of the bacterial kill among three rifamycins in the static concentration experiment. In the HFS-MAC, the bactericidal kill (day 0-4) for rifampin was 0.89 (95% Confidence Interval (CI): 0.43-1.35), for rifapentine was 1.05 (95% CI: 0.08-1.23), and for rifabutin was 0.92 (95% CI: 0.61-1.24) log10 CFU/ml, respectively. Rifamycins monotherapy failed after just 4-days of treatment and entire MAC population was drug resistant on day 26 of the study. There was no dose dependent difference in MAC kill or resistance suppression among the three rifamycins tested in the HFS-MAC. Therefore, replacing one rifamycin, due to emergence of drug-resistance, with other may not be beneficial in clinical setting.

Rickettsia rickettsii Whole-Cell Antigens Offer Protection against Rocky Mountain Spotted Fever in the Canine Host.

Rocky Mountain spotted fever (RMSF) is a potentially fatal tick-borne disease in people and dogs. RMSF is reported in the United States and several countries in North, Central, and South America. The causative agent of this disease, Rickettsia rickettsii, is transmitted by several species of ticks, including Dermacentor andersoni, Rhipicephalus sanguineus, and Amblyomma americanum RMSF clinical signs generally include fever, headache, nausea, vomiting, muscle pain, lack of appetite, and rash. If untreated, it can quickly progress into a life-threatening illness in people and dogs, with high fatality rates ranging from 30 to 80%. While RMSF has been known for over a century, recent epidemiological data suggest that the numbers of documented cases and the fatality rates remain high in people, particularly during the last two decades in parts of North America. Currently, there are no vaccines available to prevent RMSF in either dogs or people. In this study, we investigated the efficacies of two experimental vaccines, a subunit vaccine containing two recombinant outer membrane proteins as recombinant antigens (RCA) and a whole-cell inactivated antigen vaccine (WCA), in conferring protection against virulent R. rickettsii infection challenge in a newly established canine model for RMSF. Dogs vaccinated with WCA were protected from RMSF, whereas those receiving RCA developed disease similar to that of nonvaccinated R. rickettsii-infected dogs. WCA also reduced the pathogen loads to nearly undetected levels in the blood, lungs, liver, spleen, and brain and induced bacterial antigen-specific immune responses. This study provides the first evidence of the protective ability of WCA against RMSF in dogs.

Sequence determinants spanning -35 motif and AT-rich spacer region impacting Ehrlichia chaffeensis Sigma 70-dependent promoter activity of two differentially expressed p28 outer membrane protein genes.

Ehrlichia chaffeensis is an obligate intracellular tick-borne bacterium which causes the disease, human monocytic ehrlichiosis. Ehrlichia chaffeensis contains only two sigma factors, σ32 and σ70. It is difficult to study E. chaffeensis gene regulation due to lack of a transformation system. We developed an Escherichia coli-based transcription system to study E. chaffeensis transcriptional regulation. An E. coli strain with its σ70 repressed with trp promoter is used to express E. chaffeensis σ70. The E. coli system and our previously established in vitro transcription system were used to map transcriptional differences of two Ehrlichia genes encoding p28-outer membrane proteins 14 and 19. We mapped the -10 and -35 motifs and the AT rich spacers located between the two motifs by performing detailed mutational analysis. Mutations within the -35 motif of the genes impacted transcription differently, while -10 motif deletions had no impact. The AT-rich spacers also contributed to transcriptional differences. We further demonstrated that the domain 4.2 of E. chaffeensis σ70 is important for regulating promoter activity and the deletion of region 1.1 of E. chaffeensis σ70 causes enhancement of the promoter activity. This is the first study defining the promoters of two closely related E. chaffeensis genes.

Heterogeneous Associations of Ecological Attributes with Tick-Borne Rickettsial Pathogens in a Periurban Landscape.

The variations in prevalence levels of two tick-borne rickettsial pathogens, Ehrlichia chaffeensis and Ehrlichia Ewingii, in a periurban environment were evaluated along with their ecological determinants. Tick life stage and sex, month of tick collection, landscape fragmentation, and ecological covariates specific to pasture and woodland sites were considered as explanatory covariates. Questing lone star ticks (Amblyomma americanum) were collected by flagging for an hour once every week during mid-April through mid-August in years 2013 and 2014. A total of 4357 adult and nymphal ticks (woodland = 2720 and pasture = 1637) were collected and assessed for pathogen prevalence by molecular methods. Female A. americanum ticks were more infected with E. chaffeensis than males or nymphs in woodland areas [♂ = 6.05%; ♀ = 12.0%; nymphs = 2.09%] and pastures [♂ = 8.05%; ♀ = 12.03%; nymphs = 3.33%], and the prevalence was influenced by edge density in the landscape. Higher E. ewingii infection was noted among female A. americanum ticks within woodland areas [♂ = 1.89%; ♀ = 2.14%; nymphs = 1.57%], but no such difference was evident in pastures [♂ = 1.03%; ♀ = 1.33%; nymphs = 1.12%]. Prevalence of E. ewingii was influenced by edge contrast index, and the percentage of pasture perimeter that was less than 20 meters from woodland areas. This study elucidates the complexity of tick-borne pathogen ecology and points to the need for further studies on the role of reservoir hosts, particularly that played by small vertebrates, which is not fully understood in the region.