Seroepidemiologic study of human infections with spotted fever group Rickettsiae in North Carolina.

Increasing entomologic and epidemiologic evidence suggests that spotted fever group rickettsiae (SFGR) other than Rickettsia rickettsii are responsible for spotted fever rickettsioses in the United States. A retrospective seroepidemiologic study was conducted on stored acute- and convalescent-phase sera that had been submitted for Rocky Mountain spotted fever testing to the North Carolina State Laboratory of Public Health. We evaluated the serologic reactivity of the paired sera to R. rickettsii, Rickettsia parkeri, and Rickettsia amblyommii antigens. Of the 106 eligible pairs tested, 21 patients seroconverted to one or more antigens. Cross-reactivity to multiple antigens was observed in 10 patients, and seroconversions to single antigens occurred in 11 patients, including 1 against R. rickettsii, 4 against R. parkeri, and 6 against R. amblyommii. Cross-absorption of cross-reactive sera and/or Western blots identified two presumptive cases of infection with R. parkeri, two presumptive cases of infection with R. rickettsii, and one presumptive case of infection with R. amblyommii. These findings suggest that species of SFGR other than R. rickettsii are associated with illness among North Carolina residents and that serologic testing using R. rickettsii antigen may miss cases of spotted fever rickettsioses caused by other species of SFGR.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain.

BACKGROUND: The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. METHODS: To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. RESULTS: Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. CONCLUSION: Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.

Dysregulation in Genital Tract Soluble Immune Mediators in Postmenopausal Women Is Distinct by HIV Status.

A rise in new HIV diagnoses among older adults is characterized by poor prognosis and reduced survival times. Although heterosexual transmission remains the main route of infection in women, little is known regarding immune functions in the genital tract of postmenopausal women, especially those who are HIV positive. Furthermore, effects of hormone replacement therapy (HRT) on the genital tract immune system are unclear. Using the Women's Interagency HIV Study repository, we obtained cervical-vaginal lavage (CVL) samples from premenopausal and postmenopausal HIV-positive and HIV-negative women, some of whom were on HRT. Samples were assayed for interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, secretory leukocyte protease inhibitor (SLPI), Elafin, human beta defensin-2 (HBD2), and macrophage inflammatory protein (MIP)-3α using ELISA. Anti-HIV activity in CVL was measured using TZM-bl indicator cells. Among HIV-positive women, the plasma viral load was significantly higher and CD4 count was significantly lower in postmenopausal compared with premenopausal women. Postmenopausal women, irrespective of HIV status, had significantly lower levels of HBD2 compared with premenopausal women. Among the HIV-negative individuals, postmenopausal women had significantly lower levels of MIP-3α, IL-6, and SLPI compared with premenopausal women. In contrast, HIV-positive postmenopausal women had significantly higher levels of TNF-α compared with HIV-positive premenopausal women. In most cases, HRT groups resembled the postmenopausal groups. No significant differences in anti-HIV activity by menopausal or by HIV status were noted. Our findings indicate that the female genital tract immune microenvironment is distinct by menopausal status and HIV status. Further studies are needed to assess the risk of HIV acquisition/transmission in this population.

Genomic Analyses of Acute Flaccid Myelitis Cases among a Cluster in Arizona Provide Further Evidence of Enterovirus D68 Role.

Enteroviruses are a common cause of respiratory and gastrointestinal illness, and multiple subtypes, including poliovirus, can cause neurologic disease. In recent years, enterovirus D68 (EV-D68) has been associated with serious neurologic illnesses, including acute flaccid myelitis (AFM), frequently preceded by respiratory disease. A cluster of 11 suspect cases of pediatric AFM was identified in September 2016 in Phoenix, AZ. To determine if these cases were associated with EV-D68, we performed multiple genomic analyses of nasopharyngeal (NP) swabs and cerebrospinal fluid (CSF) material from the patients, including real-time PCR and amplicon sequencing targeting the EV-D68 VP1 gene and unbiased microbiome and metagenomic sequencing. Four of the 11 patients were classified as confirmed cases of AFM, and an additional case was classified as probable AFM. Real-time PCR and amplicon sequencing detected EV-D68 virus RNA in the three AFM patients from which NP swabs were collected, as well as in a fourth patient diagnosed with acute disseminated encephalomyelitis, a disease that commonly follows bacterial or viral infections, including enterovirus. No other obvious etiological causes for AFM were identified by 16S or RNA and DNA metagenomic sequencing in these cases, strengthening the likelihood that EV-D68 is an etiological factor. Herpes simplex viral DNA was detected in the CSF of the fourth case of AFM and in one additional suspect case from the cluster. Multiple genomic techniques, such as those described here, can be used to diagnose patients with suspected EV-D68 respiratory illness, to aid in AFM diagnosis, and for future EV-D68 surveillance and epidemiology.IMPORTANCE Enteroviruses frequently result in respiratory and gastrointestinal illness; however, multiple subtypes, including poliovirus, can cause severe neurologic disease. Recent biennial increases (i.e., 2014, 2016, and 2018) in cases of non-polio acute flaccid paralysis have led to speculations that other enteroviruses, specifically enterovirus D68 (EV-D68), are emerging to fill the niche that was left from poliovirus eradication. A cluster of 11 suspect cases of pediatric acute flaccid myelitis (AFM) was identified in 2016 in Phoenix, AZ. Multiple genomic analyses identified the presence of EV-D68 in the majority of clinical AFM cases. Beyond limited detection of herpesvirus, no other likely etiologies were found in the cluster. These findings strengthen the likelihood that EV-D68 is a cause of AFM and show that the rapid molecular assays developed for this study are useful for investigations of AFM and EV-D68.

Mapping and comparing bacterial microbiota in the sinonasal cavity of healthy, allergic rhinitis, and chronic rhinosinusitis subjects.

BACKGROUND: The role of microbiota in sinonasal inflammation can be further understood by targeted sampling of healthy and diseased subjects. We compared the microbiota of the middle meatus (MM) and inferior meatus (IM) in healthy, allergic rhinitis (AR), and chronic rhinosinusitis (CRS) subjects to characterize intrasubject, intersubject, and intergroup differences. METHODS: Subjects were recruited in the office, and characterized into healthy, AR, and CRS groups. Endoscopically-guided swab samples were obtained from the MM and IM bilaterally. Bacterial microbiota were characterized by sequencing the V3-V4 region of the 16S ribosomal RNA (rRNA) gene. RESULTS: Intersubject microbiome analyses were conducted in 65 subjects: 8 healthy, 11 AR, and 46 CRS (25 CRS with nasal polyps [CRSwNP]; 21 CRS without nasal polyps [CRSsNP]). Intrasubject analyses were conducted for 48 individuals (4 controls, 11 AR, 8 CRSwNP, and 15 CRSwNP). There was considerable intersubject microbiota variability, but intrasubject profiles were similar (p = 0.001, nonparametric t test). Intrasubject bacterial diversity was significantly reduced in MM of CRSsNP subjects compared to IM samples (p = 0.022, nonparametric t test). CRSsNP MM samples were enriched in Streptococcus, Haemophilus, and Fusobacterium spp. but exhibited loss of diversity compared to healthy, CRSwNP, and AR subject-samples (p < 0.05; nonparametric t test). CRSwNP patients were enriched in Staphylococcus, Alloiococcus, and Corynebacterium spp. CONCLUSION: This study presents the sinonasal microbiome profile in one of the larger populations of non-CRS and CRS subjects, and is the first office-based cohort in the literature. In contrast to healthy, AR, and CRSwNP subjects, CRSsNP MM samples exhibited decreased microbiome diversity and anaerobic enrichment. CRSsNP MM samples had reduced diversity compared to same-subject IM samples, a novel finding.

Human Infections by Multiple Spotted Fever Group Rickettsiae in Tennessee.

Rocky Mountain spotted fever is the most common tick-borne disease in Tennessee. However, Rickettsia rickettsii has rarely been isolated from endemic ticks, suggesting rickettsioses may be caused by other species. A total of 56 human serum samples that were serologically positive for exposure to Rickettsia were obtained from commercial laboratories in 2010 and 2011. In addition, 20 paired sera from patients with encephalitis and positive Rickettsia serology were obtained from the Tennessee Unexplained Encephalitis Surveillance (TUES) study. Using an immunofluorescence assay, reactivity of the sera to R. rickettsii, Rickettsia montanensis, Rickettsia parkeri, and Rickettsia amblyommii was tested, and a comparison of endpoint titers was used to determine the probable antigen that stimulated the antibody response. Cross-absorption was conducted for 94.8% (N = 91) of the samples due to serologic cross-reactivity. Of the commercial laboratory samples, 55.4% (N = 31) had specific reactivity to R. amblyommii and 44.6% (N = 25) were indeterminate. Of the paired TUES samples, 20% (N = 4) had specific reactivity to R. amblyommii, 5% (N = 1) to R. montanensis, and 5% (N = 1) to R. parkeri Patients with specific reactivity to R. amblyommii experienced fever (75%), headache (68%) and myalgia (58%). Rash (36%) and thrombocytopenia (40%) were less common. To our knowledge, this is the first time R. amblyommii has been reported as a possible causative agent of rickettsioses in Tennessee.

Development and characterization of rabbit and mouse antibodies against ebolavirus envelope glycoproteins.

Ebolaviruses are the etiologic agents of severe viral hemorrhagic fevers in primates, including humans, and could be misused for the development of biological weapons. The ability to rapidly detect and differentiate these viruses is therefore crucial. Antibodies that can detect reliably the ebolavirus surface envelope glycoprotein GP1,2 or a truncated variant that is secreted from infected cells (sGP) are required for advanced development of diagnostic assays such as sandwich ELISAs or Western blots (WB). We used a GP1,2 peptide conserved among Bundibugyo, Ebola, Reston, Sudan, and Taï Forest viruses and a mucin-like domain-deleted Sudan virus GP1,2 (SudanGPΔMuc) to immunize mice or rabbits, and developed a panel of antibodies that either cross-react or are virus-specific. These antibodies detected full-length GP1,2 and sGP in different assays such as ELISA, FACS, or WB. In addition, some of the antibodies were shown to have potential clinical relevance, as they detected ebolavirus-infected cells by immunofluorescence assay and gave a specific increase in signal by sandwich ELISA against sera from mouse-adapted Ebola virus-infected mice over uninfected mouse sera. Rabbit anti-SudanGPΔMuc polyclonal antibody neutralized gammaretroviral particles pseudotyped with Sudan virus GP1,2, but not particles pseudotyped with other ebolavirusGP1,2. Together, our results suggest that this panel of antibodies may prove useful for both in vitro analyses of ebolavirus GP1,2, as well as analysis of clinically relevant samples.

Phenylalanines at positions 88 and 159 of Ebolavirus envelope glycoprotein differentially impact envelope function.

The envelope glycoprotein (GP) of Ebolavirus (EBOV) mediates viral entry into host cells. Through mutagenesis, we and other groups reported that two phenylalanines at positions 88 and 159 of GP are critical for viral entry. However, it remains elusive which steps of viral entry are impaired by F88 or F159 mutations and how. In this study, we further characterized these two phenylalanines through mutagenesis and examined the impact on GP expression, function, and structure. Our data suggest that F159 plays an indirect role in viral entry by maintaining EBOV GP's overall structure. In contrast, we did not detect any evidence for conformational differences in GP with F88 mutations. The data suggest that F88 influences viral entry during a step after cathepsin processing, presumably impacting viral fusion.