Search

This study describes the natural history of dengue virus (DENV) infection in rhesus monkeys exposed to the bites of DENV-infected Aedes aegypti mosquitoes. Dengue virus–infected mosquitoes were generated by either intrathoracic inoculation or by oral feeding on viremic blood meals. Each of the six rhesus monkeys that were fed upon by intrathoracically infected mosquitoes developed non-structural protein 1 (NS1) antigenemia and an IgM response; viremia was detected in 4/6 individuals. No virological or immunological evidence of DENV infection was detected in the three monkeys exposed to mosquitoes that had been orally infected with DENV. These results demonstrate the utility of mosquito-borne challenge of rhesus monkeys with DENV.

Funder: Division of Intramural Research, National Institute of Allergy and Infectious Diseases, Funder: Military Infectious Disease Research Program, Funder: Armed Forces Health Surveillance Branch, Neutralizing antibodies are important correlates of protection against dengue. Yet, determinants of variation in neutralization across strains within the four dengue virus serotypes (DENV1-4) is imperfectly understood. Studies focus on structural DENV proteins, especially the envelope (E), the primary target of anti-DENV antibodies. Although changes in immune recognition (antigenicity) are often attributed to variation in epitope residues, viral processes influencing conformation and epitope accessibility also affect neutralizability, suggesting possible modulating roles of nonstructural proteins. We estimated effects of residue changes in all 10 DENV proteins on antigenic distances between 348 DENV collected from individuals living in Bangkok, Thailand (1994-2014). Antigenic distances were derived from response of each virus to a panel of twenty non-human primate antisera. Across 100 estimations, excluding 10% of virus pairs each time, 77 of 295 positions with residue variability in E consistently conferred antigenic effects; 52 were within ±3 sites of known binding sites of neutralizing human monoclonal antibodies, exceeding expectations from random assignments of effects to sites (p = 0.037). Effects were also identified for 16 sites on the stem/anchor of E which were only recently shown to become exposed under physiological conditions. For all proteins, except nonstructural protein 2A (NS2A), root-mean-squared-error (RMSE) in predicting distances between pairs held out in each estimation did not outperform sequences of equal length derived from all proteins or E, suggesting that antigenic signals present were likely through linkage with E. Adjusted for E, we identified 62/219 sites embedding the excess signals in NS2A. Concatenating these sites to E additionally explained 3.4% to 4.0% of observed variance in antigenic distances compared to E alone (50.5% to 50.8%); RMSE outperformed concatenating E with sites from any protein of the virus (ΔRMSE, 95%IQR: 0.01, 0.05). Our results support examining antigenic determinants beyond the DENV surface.

Introduction: Japanese encephalitis (JE) virus is one of the leading causes of viral encephalitis across temperate and tropical zones of Asia. The live attenuated SA 14-14-2 JE vaccine (CD-JEV) is one of three vaccines prequalified by the World Health Organization (WHO) to prevent JE. WHO currently recommends a single CD-JEV dose for infants in endemic settings. However, in the absence of long-term immunogenicity data, WHO has indicated a need for long-term immunogenicity studies to inform optimal dosing schedules and determine the need for booster doses. Methods: This Phase 4, open-label clinical study measured neutralizing antibody (NAb) titers in Bangladeshi children three and four years after primary CD-JEV vaccination and 7 and 28 days after a booster CD-JEV vaccination given four years after primary vaccination. The study also assessed the tolerability and safety of the booster dose. A NAb titer of ≥1:10 was considered seroprotective. Results: Of 560 children vaccinated between 10 and 12 months of age with CD-JEV three years earlier and enrolled in this study from 30 July 2015 through 03 January 2016, 52 (9.3%; 95% CI: 7.2–12.0) had a seroprotective titer at enrollment. One year later, of 533 children, 66 (12.4%; 95% CI: 9.9–15.5) had a seroprotective titer before receiving a booster dose. Of 524 children who received a booster CD-JEV dose, 479 (91.4%; 95% CI: 88.7–93.5) and 514 (98.1%; 95% CI: 96.5–99.0) were seroprotected 7 and 28 days later, respectively. The geometric mean titer (GMT) was 6 (95% CI: 6–6) at baseline, 105 (95% CI: 93–119) 7 days post-booster, and 167 (95% CI: 152–183) 28 days post-booster. No vaccine-associated neurologic adverse events or other serious adverse events were noted following the booster dose. Conclusions: Although most children did not have measurable antibody titers three and four years after a single primary CD-JEV dose, more than 90% of seronegative children had a strong anamnestic response within one week of a booster dose. This suggests that these children were immune despite the absence of measurable NAb prior to their booster.ClinicalTrials.gov Identifier: NCT02514746.

Despite recommendations, few children aged 6–35 months in Thailand receive seasonal influenza vaccination. Using previously estimated incidence and vaccine effectiveness data from the period 2012–2014, we estimate that up to 121 000 medical visits could be prevented each year with 50% coverage and expanded recommendations to children aged

Difficulties inherent in the identification of immune correlates of protection or severe disease have challenged the development and evaluation of dengue vaccines. There persist substantial gaps in knowledge about the complex effects of age and sequential dengue virus (DENV) exposures on these correlations. To address these gaps, we were conducting a novel family-based cohort-cluster study for DENV transmission in Kamphaeng Phet, Thailand. The study began in 2015 and is funded until at least 2023. As of May 2019, 2,870 individuals in 485 families were actively enrolled. The families comprise at least 1 child born into the study as a newborn, 1 other child, a parent, and a grandparent. The median age of enrolled participants is 21 years (range 0–93 years). Active surveillance is performed to detect acute dengue illnesses, and annual blood testing identifies subclinical seroconversions. Extended follow-up of this cohort will detect sequential infections and correlate antibody kinetics and sequence of infections with disease outcomes. The central goal of this prospective study is to characterize how different DENV exposure histories within multigenerational family units, from DENV-naive infants to grandparents with multiple prior DENV exposures, affect transmission, disease, and protection at the level of the individual, household, and community.

The use of next generation sequencing (NGS) directly on respiratory specimens to obtain viral whole genome sequence (WGS) enhances the capability for rapid and unbiased viral characterization. One of the challenges of using NGS directly in influenza-like illness (ILI) respiratory specimens is the higher proportion of host and bacterial genome compared to viral genetic materials found, which reduces the likelihood of obtaining complete viral genome sequences. This study aims to evaluate nuclease pretreatments prior to sequencing of influenza B virus directly from ILI respiratory specimens. Sequence data were mapped to human, bacteria and influenza B viral genome. In the absence of any nuclease pretreatments, the sequence reads identified as Haemophilus influenzae, Haemophilus parainfluenzae, Neisseria meningitidis and Veillonella parvula were the most prominent genetic materials in respiratory specimens. Filtration followed by nuclease treatment reduced bacterial sequence reads by at least 70 folds in all 4 tested samples, supporting the direct application of NGS in ILI respiratory specimens. Although the pretreatment methods significantly reduced human genome sequences, the remaining human genome especially human rRNA still impact the number and proportion of the viral sequence reads.

Highlights • Live, attenuated SA 14-14-2 JE vaccine is immunogenic when given with MMR vaccine. • Live, attenuated SA 14-14-2 JE vaccine is safe when given with MMR vaccine. • Live, attenuated SA 14-14-2 JE vaccine may be coadministered with MMR vaccine., Introduction Japanese encephalitis (JE) virus is the leading cause of viral encephalitis across temperate and tropical zones of Asia. The live attenuated SA 14-14-2 JE vaccine (CD-JEV) is one of three vaccines prequalified by the World Health Organization (WHO) to prevent JE. When incorporating a new vaccine into a country’s Expanded Program on Immunization (EPI), it is important to show that the new vaccine can be administered concurrently with other routine pediatric vaccines without impairing the immune responses or changing the safety profiles of the co-administered vaccines. This Phase 4 open-label study evaluated the safety and immunogenicity of measles-mumps-rubella (MMR) vaccine co-administered with CD-JEV. Methods The study randomized 628 healthy Filipino children aged between 9 and 10 months to receive MMR and CD-JEV concurrently or separately. MMR immunogenicity was measured 56 days after MMR vaccination using a measles plaque reduction neutralization test (PRNT), anti-mumps immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), and anti-rubella IgG ELISA, respectively. Neutralizing antibody against JE virus was measured 28 days after CD-JEV vaccination using PRNT. Safety was assessed through solicitation of immediate reactions, adverse events (AEs) within 14 days of vaccination, unsolicited AEs occurring within 28 days, and serious adverse events (SAEs) during participation in the study. Results/Conclusions During the study, no post-vaccinal encephalitis cases or related SAEs were reported in either group. Concurrent immunization with CD-JEV and MMR vaccines was not associated with any unusual safety signals when compared with sequential immunization. No significant differences between the regimens were seen in seropositivity or serology titer/concentration results for any of the antigens. Co-administration of CD-JEV and MMR was non-inferior to single administration of either vaccine.

Military recruits are at high risk of respiratory infections.1 The congregation of individuals from diverse geographic locations in semi-closed settings, together with high levels of close contact, provide conditions that favor the introduction and transmission of respiratory pathogens.2 Studies among military recruits have found high rates of illness and infection with respiratory viruses. A longitudinal study in Singapore found that 39.4% of military personnel had evidence of seroconversion against influenza A/H1N1 during the 2009 pandemic, compared with 13.5% of individuals in the community and 6.5% of hospital staff.3 Numerous outbreaks of respiratory infections among military recruits have been reported in the literature, including outbreaks of influenza, adenovirus,4–6 and pertussis.7 Resumption of vaccination against adenovirus types 4 and 7 in the U.S. military is estimated to have resulted in a 7-fold decrease in acute respiratory disease8 and a shift in the predominant adenovirus types from types 4, 3, and 7 to types 1 and 2. The increasing availability of multiplex molecular diagnostic assays can yield data on multiple pathogens that would not routinely be detected.9,10 Studies in military populations can provide valuable information regarding the epidemiology and transmission of respiratory infections in adults because of the availability of well-defined populations that can be followed up over time. Despite this, data from tropical settings, in which the epidemiology of influenza and other respiratory infections differs substantially from temperate settings, are limited. Increased understanding of the etiology of respiratory infections in these settings is important to identify opportunities for disease control in high-risk military populations and to identify risks of pathogen emergence with potential for spread into the wider community. We present the results of a longitudinal study of the burden and etiology of respiratory infections in Thai military recruits.

Influenza is an infectious respiratory illness caused by influenza viruses. Despite yearly updates, the efficacy of influenza vaccines is significantly curtailed by the virus antigenic drift and antigenic shift. These constant changes to the influenza virus make-up also challenge the development of a universal flu vaccine, which requires conserved antigenic regions shared by influenza viruses of different subtypes. We propose that it is possible to bypass these challenges by the development of an influenza vaccine based on conserved proteins delivered in an adjuvanted nanoparticle system. In this study, we generated influenza nanoparticle constructs using trimethyl chitosan nanoparticles (TMC nPs) as the carrier of recombinant influenza hemagglutinin subunit 2 (HA2) and nucleoprotein (NP). The purified HA2 and NP recombinant proteins were encapsulated into TMC nPs to form HA2-TMC nPs and NP-TMC nPs, respectively. Primary human intranasal epithelium cells (HNEpCs) were used as an in vitro model to measure immunity responses. HA2-TMC nPs, NP-TMC nPs, and HA2-NP-TMC nPs (influenza nanoparticle constructs) showed no toxicity in HNEpCs. The loading efficiency of HA2 and NP into the TMC nPs was 97.9% and 98.5%, respectively. HA2-TMC nPs and NP-TMC nPs more efficiently delivered HA2 and NP proteins to HNEpCs than soluble HA2 and NP proteins alone. The induction of various cytokines and chemokines was more evident in influenza nanoparticle construct-treated HNEpCs than in soluble protein-treated HNEpCs. In addition, soluble factors secreted by influenza nanoparticle construct-treated HNEpCs significantly induced MoDCs maturation markers (CD80, CD83, CD86 and HLA-DR), as compared to soluble factors secreted by protein-treated HNEpCs. HNEpCs treated with the influenza nanoparticle constructs significantly reduced influenza virus replication in an in vitro challenge assay. The results indicate that TMC nPs can be used as influenza vaccine adjuvants and carriers capable of delivering HA2 and NP proteins to HNEpCs.

Prior exposure to dengue virus (DENV) has a profound impact on the outcome of infection, which varies according to the interval between infections. Antibodies secreted by B cells and cytokines secreted by T cells are thought to contribute both to protective immunity against DENV and the pathogenesis of dengue disease. We analyzed peripheral blood mononuclear cells (PBMC) collected from Thai children over a 5-year prospective cohort study to define the dynamics of DENV-specific memory B and T cell responses and the impact of symptomatic or subclinical DENV infections. To measure B cell responses, PBMC were stimulated with IL-2 plus R848 and culture supernatants were tested for DENV-binding antibodies by ELISA. To measure T cell responses, PBMC were stimulated in dual-color ELISPOT assays with overlapping peptide pools of structural and non-structural proteins from the four DENV types. B cell responses were low to one or more DENV types prior to symptomatic infection and increased with reactivity to all four types after infection. Subjects who had a subclinical infection or who did not experience a DENV infection during the study period showed strong memory B cell responses to all four DENV types. T cell responses to DENV peptides demonstrated a cytokine hierarchy of IFN-γ > IL-2 > IFN-γ/IL-2. T cell responses were low or absent prior to secondary infections. The trends in T cell responses to DENV peptides over 3 year post-infection were highly variable, but subjects who had experienced a secondary DENV1 infection showed higher cytokine responses compared to subjects who had experienced a secondary DENV2 or subclinical infection. The longitudinal nature of our study demonstrates persistent memory B cell responses over years and a lasting but variable impact of secondary DENV infection on DENV-specific T cell responses.

Background Multiplex real-time polymerase chain reaction assays have improved diagnostic sensitivity for a wide range of pathogens. However, co-detection of multiple agents and bacterial colonization make it difficult to distinguish between asymptomatic infection or illness aetiology. We assessed whether semi-quantitative microbial load data can differentiate between symptomatic and asymptomatic states for common respiratory pathogens. Methods We obtained throat and nasal swab samples from military trainees at two Thai Army barracks. Specimens were collected at the start and end of 10-week training periods (non-acute samples), and from individuals who developed upper respiratory tract infection during training (acute samples). We analysed the samples using a commercial multiplex respiratory panel comprising 33 bacterial, viral and fungal targets. We used random effects tobit models to compare cycle threshold (Ct) value distributions from non-acute and acute samples. Results We analysed 341 non-acute and 145 acute swab samples from 274 participants. Haemophilus influenzae type B was the most commonly detected microbe (77.4% of non-acute and 64.8% of acute samples). In acute samples, nine specific microbe pairs were detected more frequently than expected by chance. Regression models indicated significantly lower microbial load in non-acute relative to acute samples for H. influenzae non-type B, Streptococcus pneumoniae and rhinovirus, although it was not possible to identify a Ct-value threshold indicating causal etiology for any of these organisms. Conclusions Semi-quantitative measures of microbial concentration did not reliably differentiate between illness and asymptomatic colonization, suggesting that clinical symptoms may not always be directly related to microbial load for common respiratory infections. Electronic supplementary material The online version of this article (10.1186/s12879-018-3358-4) contains supplementary material, which is available to authorized users.

Early diagnosis of influenza infection maximizes the effectiveness of antiviral medicines. Here, we assess the ability for clinical characteristics and rapid influenza tests to predict PCR-confirmed influenza infection in a sentinel, cross-sectional study for influenza-like illness (ILI) in Thailand. Participants meeting criteria for acute ILI (fever > 38°C and cough or sore throat) were recruited from inpatient and outpatient departments in Bangkok, Thailand, from 2009–2014. The primary endpoint for the study was the occurrence of virologically-confirmed influenza infection (based upon detection of viral RNA by RT-PCR) among individuals presenting for care with ILI. Nasal and throat swabs were tested by rapid influenza test (QuickVue) and by RT-PCR. Vaccine effectiveness (VE) was calculated using the case test-negative method. Classification and Regression Tree (CART) analysis was used to predict influenza RT-PCR positivity based upon symptoms reported. We enrolled 4572 individuals with ILI; 32.7% had detectable influenza RNA by RT-PCR. Influenza cases were attributable to influenza B (38.6%), A(H1N1)pdm09 (35.1%), and A(H3N2) (26.3%) viruses. VE was highest against influenza A(H1N1)pdm09 virus and among adults. The most important symptoms for predicting influenza PCR-positivity among patients with ILI were cough, runny nose, chills, and body aches. The accuracy of the CART predictive model was 72.8%, with an NPV of 78.1% and a PPV of 59.7%. During epidemic periods, PPV improved to 68.5%. The PPV of the QuickVue assay relative to RT-PCR was 93.0% overall, with peak performance during epidemic periods and in the absence of oseltamivir treatment. Clinical criteria demonstrated poor predictive capability outside of epidemic periods while rapid tests were reasonably accurate and may provide an acceptable alternative to RT-PCR testing in resource-limited areas.

Background Influenza infection in children can be severe, resulting in complications such as pneumonia, but may be mitigated by early recognition and administration of antivirals. In this study, we identified risk factors for hospitalization and pneumonia in a pediatric population presenting with influenza-like illness (ILI) in Thailand. Methods Our study included pediatric patients (age < 18 years) presenting with ILI to inpatient and outpatient departments at a public hospital in Bangkok, Thailand, from 2009 to 2016. ILI was defined as fever plus cough or sore-throat, and pneumonia was defined as either lung radiographic or pulmonary examination abnormalities. Demographic and clinical data, as well as nasal and throat swabs, were collected during a one-time interview with patients presenting with ILI. Influenza infections were confirmed via RT-PCR testing of respiratory specimens. Retrospective chart review was used to collect data on individuals with influenza admitted for inpatient care. Results 5,968 children (33.6%) were enrolled with ILI, of whom 1,530 (25.6%) were confirmed to be influenza by RT-PCR, of which 25.5% were influenza A(H1N1)pdm09, 31.5% influenza A(H3N2), and 43.0% influenza B. 124 (8.1%) patients were admitted, and 41 of these children (33.1%) developed pneumonia. Predictors of hospitalization included younger age (4.1 yrs for inpatients vs. 5.6 yrs) and higher presenting temperature (38.6C for inpatient vs. 38.0C) (both P < 0.05). Among children hospitalized with influenza, influenza subtype was not associated with pneumonia risk. Co-detection of Klebsiella pneumoniae was associated with an increased risk of pneumonia (P < 0.05. Patients with pneumonia were younger (4.1 yrs with vs. 6.4 yrs, P = NS), had a longer interval from fever onset to presentation at the hospital, and required longer hospital stays. Risk of pneumonia was decreased in patients who received oseltamivir within 48 hours of fever onset (odds ratio 0.36, 95% confidence interval 0.16–0.91). Conclusion Post viral pneumonia is a potentially serious complication of influenza, requiring longer hospitalization stay and affecting more than one-third of hospitalized pediatric patients with influenza. The risk of pneumonia can be reduced with early presentation for clinical care and prompt administration of oseltamivir following fever onset. Disclosures All authors: No reported disclosures.

Spotted fever group rickettsiae are known to produce distinct plaque phenotypes. Strains that cause lytic infections in cell culture form clear plaques, while nonlytic strains form opaque plaques in which the cells remain intact. Clear plaques have historically been associated with more-virulent species or strains of spotted fever group rickettsiae. We have selected spontaneous mutant pairs from two independent strains of Rickettsia rickettsii , the virulent R strain and the avirulent Iowa strain. A nonlytic variant of R. rickettsii R, which typically produces clear plaques, was isolated and stably maintained. A lytic variant of the Iowa strain, which characteristically produces opaque plaques, was also selected and maintained. Genomic resequencing of the variants identified only a single gene disrupted in each strain. In both cases, the mutation was in a gene annotated as relA / spoT -like. In the Iowa strain, a single mutation introduced a premature stop codon upstream from region encoding the predicted active site of RelA/SpoT and caused the transition to a lytic plaque phenotype. In R. rickettsii R, the nonlytic plaque phenotype resulted from a single-nucleotide substitution that shifted a tyrosine residue to histidine near the active site of the enzyme. The intact relA / spoT gene thus occurred in variants with the nonlytic plaque phenotype. Complementation of the truncated relA / spoT gene in the Iowa lytic plaque variant restored the nonlytic phenotype. The relA / spoT mutations did not affect the virulence of either strain in a Guinea pig model of infection; R strain lytic and nonlytic variants both induced fever equally, and the mutation in Iowa to a lytic phenotype did not cause them to become virulent.

Rickettsii rickettsii, the etiologic agent of Rocky Mountain spotted fever, replicates within the cytosol of infected cells and uses actin-based motility to spread inter- and intracellularly. Although the ultrastructure of the actin tail and host proteins associated with it are distinct from those ofListeriaorShigella, comparatively little is known regarding the rickettsial proteins involved in its organization. Here, we have used random transposon mutagenesis ofR. rickettsiito generate a small-plaque mutant that is defective in actin-based motility and does not spread directly from cell to cell as is characteristic of spotted fever group rickettsiae. The transposon insertion site of this mutant strain was within Sca2, a member of a family of large autotransporter proteins. Sca2 exhibits several features suggestive of its apparent role in actin-based motility. It displays an N-terminal secretory signal peptide, a C-terminal predicted autotransporter domain, up to four predicted Wasp homology 2 (WH2) domains, and two proline-rich domains, one with similarity to eukaryotic formins. In a guinea pig model of infection, the Sca2 mutant did not elicit fever, suggesting that Sca2 and actin-based motility are virulence factors of spotted fever group rickettsiae.

inv encodes invasin, which is the primary invasion factor of Yersinia enterocolitica. inv expression in vitro is regulated in response to temperature, pH, and growth phase. In vitro, inv is maximally expressed at 26°C and repressed at 37°C at neutral pH but, when the pH of the media is adjusted to 5.5, levels of inv expression at 37°C are comparable to those at 26°C. A previous genetic screen for regulators of inv identified RovA, which was found to be required for activation of inv in vitro under all conditions tested as well as in vivo. Here we describe a screen that has identified a negative regulator of inv expression, ymoA . The ymoBA locus was identified by transposon mutagenesis as a repressor of inv expression in vitro at 37°C at neutral pH. This mutant shows increased inv expression at 37°C. The mutant can be fully complemented for inv expression by a plasmid expressing ymoA . These results indicate that YmoA plays a role in the negative regulation of inv .

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. Strains of R. rickettsii differ dramatically in virulence. In a guinea pig model of infection, the severity of disease as assessed by fever response varies from the most virulent, Sheila Smith, to Iowa, which causes no fever. To identify potential determinants of virulence in R. rickettsii , the genomes of two additional strains were sequenced for comparison to known sequences (comparative genome sequencing [CGS]). R. rickettsii Morgan and R strains were compared to the avirulent R. rickettsii Iowa and virulent R. rickettsii Sheila Smith strains. The Montana strains Sheila Smith and R were found to be highly similar while the eastern strains Iowa and Morgan were most similar to each other. A major surface antigen, rickettsial outer membrane protein A (rOmpA), is severely truncated in the Iowa strain. The region of ompA containing 13 tandem repeats was sequenced, revealing only seven shared SNPs (four nonsynonymous) for R and Morgan strains compared to Sheila Smith, with an additional 17 SNPs identified in Morgan. Another major surface antigen and autotransporter, rOmpB, exhibits a defect in processing in the Iowa strain such that the beta fragment is not cleaved. Sequence analysis of ompB reveals identical sequences between Iowa and Morgan strains and between the R and Sheila Smith strains. The number of SNPs and insertions/deletions between sequences of the two Montana strains and the two eastern strains is low, thus narrowing the field of possible virulence factors.

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. To identify genes involved in the virulence of R. rickettsii , the genome of an avirulent strain, R. rickettsii Iowa, was sequenced and compared to the genome of the virulent strain R. rickettsii Sheila Smith. R. rickettsii Iowa is avirulent in a guinea pig model of infection and displays altered plaque morphology with decreased lysis of infected host cells. Comparison of the two genomes revealed that R. rickettsii Iowa and R. rickettsii Sheila Smith share a high degree of sequence identity. A whole-genome alignment comparing R. rickettsii Iowa to R. rickettsii Sheila Smith revealed a total of 143 deletions for the two strains. A subsequent single-nucleotide polymorphism (SNP) analysis comparing Iowa to Sheila Smith revealed 492 SNPs for the two genomes. One of the deletions in R. rickettsii Iowa truncates rompA , encoding a major surface antigen (rickettsial outer membrane protein A [rOmpA]) and member of the autotransporter family, 660 bp from the start of translation. Immunoblotting and immunofluorescence confirmed the absence of rOmpA from R. rickettsii Iowa. In addition, R. rickettsii Iowa is defective in the processing of rOmpB, an autotransporter and also a major surface antigen of spotted fever group rickettsiae. Disruption of rompA and the defect in rOmpB processing are most likely factors that contribute to the avirulence of R. rickettsii Iowa. Genomic differences between the two strains do not significantly alter gene expression as analysis of microarrays revealed only four differences in gene expression between R. rickettsii Iowa and R. rickettsii strain R. Although R. rickettsii Iowa does not cause apparent disease, infection of guinea pigs with this strain confers protection against subsequent challenge with the virulent strain R. rickettsii Sheila Smith.

RovA is a transcriptional activator of Yersinia invasin, an outer membrane protein involved in bacterial attachment and invasion across the intestinal epithelium. In Y. enterocolitica, a rovA mutant is attenuated for virulence compared with either wild-type or inv mutant strains, indicating that RovA may regulate additional virulence factors. Here, we used microarray analysis to define the RovA regulon. Curiously, there was little overlap between the RovA regulons of Y. enterocolitica and Y. pestis despite the fact that RovA itself is highly conserved between the two species. Some of these differences are explained by the observation that a number of RovA-regulated loci in Y. enterocolitica do not have orthologues in Y. pestis and vice versa, suggesting that RovA established regulatory control over genetic material acquired after the divergence of the species. Electromobility shift assays demonstrated that 15 of these RovA-regulated loci directly interact with RovA, and 11 of these promoters had similar affinity as observed for the inv promoter. H-NS and YmoA are believed to form a transcriptional repression complex on the inv promoter, and several studies indicate that RovA and H-NS have overlapping DNA binding sites. H-NS and YmoA regulated a subset of the RovA-regulated loci. Furthermore, H-NS directly bound to 14 of the 15 promoters bound by RovA. From these data, we hypothesize that RovA generally behaves as an anti-H-NS factor to alleviate transcriptional repression in Y. enterocolitica. A number of recent studies have presented data and a model suggesting that H-NS functions as a transcriptional silencer of horizontally acquired genes. This repression can be selectively relieved by regulators such as RovA, and the observation that nearly all RovA-activated genes are repressed by H-NS is consistent with this model.

Virulence gene regulators RovA, SlyA and PecS comprise a subset of the MarR/SlyA family of transcriptional regulators, which has been shown to be involved in the regulation of virulence genes. These regulators have all been shown to both positively and negatively regulate the expression of multiple genes, involving several different mechanisms. One of the conserved mechanisms of regulatory control among these proteins appears to be competition for binding sites with other proteins. SlyA negatively regulates its own expression by interfering with the binding of RNA polymerase, whereas RovA appears to interfere with the progression of RNA polymerase from its promoter and to compete for binding with the heat-stable nucleoid-structural protein (H-NS), a global transcriptional silencer. PecS represses transcription by competing for binding with cAMP receptor protein, a global activator. RovA, SlyA and PecS have all been shown to act as derepressors by competing for binding sites with repressors. Recently, RovA also was found to enhance transcription through interaction with RNA polymerase.

RovA, a member of the MarR/SlyA family of winged-helix transcription factors, regulates expression of invasin, the major adhesion and invasion factor in Yersinia enterocolitica and Yersinia pseudotuberculosis. Disruption of rovA increases the LD(50) of the organism when inoculated using the oral route. However, when administered by intraperitoneal injection only a slight difference in LD(50) between mutant and wild-type organisms is apparent. The study of RovA and the genes it regulates provides a unique opportunity to gain insight into the initial stages of a Yersinia infection.

PhoB is the response regulator of the Pho regulon. It is composed of two distinct domains, an N-terminal receiver domain and a C-terminal output domain that binds DNA and interacts with ς 70 to activate transcription of the Pho regulon. Phosphorylation of the receiver domain is required for activation of the protein. The mechanism of activation by phosphorylation has not yet been determined. To better understand the function of the receiver domain in controlling the activity of the output domain, a direct comparison was made between unphosphorylated PhoB and its solitary DNA-binding domain (PhoB DBD ) for DNA binding and transcriptional activation. Using fluorescence anisotropy, it was found that PhoB DBD bound to the pho box with an affinity seven times greater than that of unphosphorylated PhoB. It was also found that PhoB DBD was better able to activate transcription than the full-length, unmodified protein. We conclude that the unphosphorylated receiver domain of PhoB silences the activity of its output domain. These results suggest that upon phosphorylation of the receiver domain of PhoB, the inhibition placed upon the output domain is relieved by a conformational change that alters interactions between the unphosphorylated receiver domain and the output domain.