Mark J. Delorey

Humoral immune responses of dengue fever patients using epitope-specific serotype-2 virus-like particle antigens.

Dengue virus (DENV) is a serious mosquito-borne pathogen causing significant global disease burden, either as classic dengue fever (DF) or in its most severe manifestation dengue hemorrhagic fever (DHF). Nearly half of the worlds population is at risk of dengue disease and there are estimated to be millions of infections annually; a situation which will continue to worsen with increasing expansion of the mosquito vectors and epidemic DF/DHF. Currently there are no available licensed vaccines or antivirals for dengue, although significant effort has been directed toward the development of safe and efficacious dengue vaccines for over 30 years. Promising vaccine candidates are in development and testing phases, but a better understanding of immune responses to DENV infection and vaccination is needed. Humoral immune responses to DENV infection are complex and may exacerbate pathogenicity, yet are essential for immune protection. In this report, we develop DENV-2 envelope (E) protein epitope-specific antigens and measure immunoglobulin responses to three distinct epitopes in DENV-2 infected human serum samples. Immunoglobulin responses to DENV-2 infection exhibited significant levels of individual variation. Antibody populations targeting broadly cross-reactive epitopes centered on the fusion peptide in structural domain II were large, highly variable, and greater in primary than in secondary DENV-2 infected sera. E protein domain III cross-reactive immunoglobulin populations were similarly variable and much larger in IgM than in IgG. DENV-2 specific domain III IgG formed a very small proportion of the antibody response yet was significantly correlated with DENV-2 neutralization, suggesting that the highly protective IgG recognizing this epitope in murine studies plays a role in humans as well. This report begins to tease apart complex humoral immune responses to DENV infection and is thus important for improving our understanding of dengue disease and immunological correlates of protection, relevant to DENV vaccine development and testing.

Incidence of Clinician-Diagnosed Lyme Disease, United States, 2005-2010.

Extrapolation from a large medical claims database suggests that 329,000 cases occur annually.

Comparison of Two Commercially Available Dengue Virus (DENV) NS1 Capture Enzyme-Linked Immunosorbent Assays Using a Single Clinical Sample for Diagnosis of Acute DENV Infection

ABSTRACT Dengue virus (DENV) nonstructural protein 1 (NS1) has shown promise as a novel diagnostic marker of acute DENV infection. Current techniques used to diagnose acute DENV infection, including virus isolation and reverse transcription-PCR (RT-PCR), are costly and difficult to perform, while traditional serological assays have low sensitivities during the acute stage of infection. Two commercially available NS1 antigen capture enzyme-linked immunosorbent assays (ELISAs), the Platelia dengue NS1Ag test (Bio-Rad Laboratories, Marnes La Coquette, France) and the Pan-E dengue early ELISA test (Panbio Diagnostics, Brisbane, Australia), were evaluated against a well-characterized panel of 208 real-time RT-PCR- and virus isolation-positive sera, as well as 45 real-time RT-PCR- and serologically negative sera from patients with other acute febrile illnesses. The overall sensitivities were 64.9% (95% confidence interval [CI95], 58.2 to 71.1%) for the Panbio test and 83.2% (CI95, 77.5 to 87.7%) for the Bio-Rad test, with interserotype variation, especially for DENV serotype 4. Predictive models were constructed to identify factors that had a significant influence on a tests outcome with respect to this panel of samples in order to identify the conditions in which the test will be most effective as a diagnostic tool. The immunoglobulin G titer was found to be the only covariate that significantly influenced results in the Bio-Rad test, while serotype and the day postonset were found to significantly influence results in the Panbio test. We concluded that the NS1 capture ELISA is a useful tool that can improve testing algorithms to diagnose DENV infection in single samples from acute and early convalescent cases.

West Nile Virus RNA Not Detected in Urine of 40 People Tested 6 Years After Acute West Nile Virus Disease

West Nile virus (WNV) causes an acute infection that is usually cleared by an effective immune response after several days of viremia. However, a recent study detected WNV RNA in the urine of 5 of 25 persons (20%) tested several years after their initial acute WNV disease. We evaluated an established cohort of 40 persons >6 years after initial infection with WNV. Urine collected from all participants tested negative for WNV RNA by reverse-transcription polymerase chain reaction and transcription-mediated amplification. Prospective studies are needed to determine if and for how long WNV persists in urine following WNV disease.

Virulence differences among Francisella tularensis subsp. tularensis clades in mice.

Francisella tularensis subspecies tularensis (type A) and holarctica (type B) are of clinical importance in causing tularemia. Molecular typing methods have further separated type A strains into three genetically distinct clades, A1a, A1b and A2. Epidemiological analyses of human infections in the United States suggest that A1b infections are associated with a significantly higher mortality rate as compared to infections caused by A1a, A2 and type B. To determine if genetic differences as defined by molecular typing directly correlate with differences in virulence, A1a, A1b, A2 and type B strains were compared in C57BL/6 mice. Here we demonstrate significant differences between survival curves for infections caused by A1b versus A1a, A2 and type B, with A1b infected mice dying earlier than mice infected with A1a, A2 or type B; these results were conserved among multiple strains. Differences were also detected among type A clades as well as between type A clades and type B with respect to bacterial burdens, and gross anatomy in infected mice. Our results indicate that clades defined within F. tularensis subsp. tularensis by molecular typing methods correlate with virulence differences, with A1b strains more virulent than A1a, A2 and type B strains. These findings indicate type A strains are not equivalent with respect to virulence and have important implications for public health as well as basic research programs.

Utility of a Commercial Nonstructural Protein 1 Antigen Capture Kit as a Dengue Virus Diagnostic Tool

ABSTRACT Annually, over 2.5 billion people are at risk for infection with dengue virus (DENV), while between 50 and 100 million people contract the infection. There is an urgent need for alternative diagnostic tools that can detect DENV during acute infection. Recent studies have shown that DENV nonstructural protein 1 (NS1) is detectable in the blood as early as the onset of symptoms and persists well into the convalescent phase of the infection. We evaluated the utility of the Bio-Rad Platelia DENV NS1 antigen capture kit in combination with real-time reverse transcriptase PCR (RT-PCR) and an IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for refining a new algorithm for the diagnosis of acute- or convalescent-phase DENV infection with a single clinical sample. We tested the Bio-Rad kit with three panels of sera. These panels were designed to evaluate the sensitivities of the NS1 kit for (i) early-convalescent-phase samples, (ii) acute-phase samples with false-negative PCR results, and (iii) IgM-negative convalescent-phase samples from patients with confirmed secondary DENV infections. Results show that NS1 can be detected in 22% of serum samples collected more than 10 days after the onset of illness and in 22% of samples that did not elicit an IgM response. Additionally, NS1 was detected in 37% of the tested acute-phase samples with false-negative PCR results, suggesting that NS1 detection may be valuable in increasing the sensitivity of current acute-phase diagnostics. These results will improve diagnosis with a single acute-phase or early-convalescent-phase sample for disease surveillance and clinical diagnosis.

Host-Seeking Heights, Host-Seeking Activity Patterns, and West Nile Virus Infection Rates for Members of the Culex pipiens Complex at Different Habitat Types Within the Hybrid Zone, Shelby County, TN, 2002 (Diptera: Culicidae)

Abstract Host-seeking heights, host-seeking activity patterns, and West Nile virus (family Flaviviridae, genus Flavivirus, WNV) infection rates were assessed for members of the Culex pipiens complex from July to December 2002, by using chicken-baited can traps (CT) at four ecologically different sites in Shelby County, TN. Host-seeking height was assessed by CT placed at elevations of 3.1, 4.6, and 7.6 m during one 24-h period per month. Host-seeking activity was assessed by paired CT placed at an elevation of 4.6 m. Can traps were sampled at one 10-h daytime interval and at seven 2-h intervals during the evening, night, and morning. Cx. pipiens complex mosquitoes accounted for 87.1% of collected mosquitoes. Culex (Melanoconion) erraticus (Dyar & Knab) accounted for 11.9% of specimens. The average number of Cx. pipiens complex mosquitoes collected per 24-h CT period from July to September was lowest at a rural middle income site (1.7), intermediate at an urban middle income site (11.3), and highest at an urban low income site (47.4). Can traps at the forested site failed to collect Cx. pipiens complex mosquitoes. From July to September at urban sites, Culex pipiens pipiens L. was the rarest of the three complex members accounting for 11.1–25.6% of specimens. At the rural site, Culex pipiens quinquefasciatus Say was the rarest member of the complex. Cx. p. pipiens was not collected after September. Mean abundance of Cx. pipiens complex mosquitoes was higher in traps at 7.6 m than in traps at 4.6 m. Abundances at 3.1 m were intermediate and not significantly different from abundances at the other heights. Initiation of host-seeking activity was associated with the end of civil twilight and activity occurred over an extended nighttime period lasting 8–10 h. All 11 WNV-positive mosquitoes were Cx. pipiens complex mosquitoes collected from urban sites in traps placed at elevations of 4.6 and 7.6 m. Infection rates were marginally nonsignificant by height. Infection rates, host-seeking heights, and activity patterns were not significantly different among members of the Cx. pipiens complex.

Oviposition Activity Patterns and West Nile Virus Infection Rates for Members of the Culex pipiens Complex at Different Habitat Types within the Hybrid Zone, Shelby County, TN, 2002 (Diptera: Culicidae)

Abstract Oviposition activity and West Nile virus (family Flaviviridae, genus Flavivirus, WNV) infection rates were assessed for members of the Culex pipiens complex from July through December 2002 by using gravid traps placed at four ecologically different sites in the southern portion of the hybrid zone in Shelby County, TN. Molecular assays identified three members of the Cx. pipiens complex: Cx. pipiens pipiens L., Cx. p. quinquefasciatus Say, and Cx. p. pipiens–Cx. p. quinquefasciatus hybrids (hybrids). The Cx. pipiens complex accounted for 90% of mosquitoes collected in gravid traps. All 285 WNV-positive mosquitoes were Culex mosquitoes, and 277 (97%) were Cx. pipiens complex mosquitoes. Infection rates among members of the Cx. pipiens complex were not significantly different. Infection rates were significantly higher at two urban sites than at a rural site, and WNV was not detected at a forested site. At urban sites, abundances of members of the Cx. pipiens complex corresponded to a simple latitude model of the hybrid zone. Cx. p. quinquefasciatus was most abundant (46.4%), followed by hybrids (34.1%) and Cx. p. pipiens (19.5%). The relative abundances at a rural site were reversed with Cx. p. pipiens (48.4%) being most abundant. This demonstrates that spatial habitat variation may profoundly influence the distribution of members of the Cx. pipiens complex within the hybrid zone. Members of the Cx. pipiens complex did not display different oviposition patterns. However, oviposition patterns assessed hourly at urban and rural sites were significantly different. At urban sites, oviposition activity of Cx. pipiens complex mosquitoes was bimodal with an evening peak associated with sunset and a morning peak associated with sunrise. At the rural site, the evening peak was pronounced and the morning peak weak and similar to nighttime activity.

Evaluation of Chimeric Japanese Encephalitis and Dengue Viruses for Use in Diagnostic Plaque Reduction Neutralization Tests

ABSTRACT The plaque reduction neutralization test (PRNT) is a specific serological test used to identify and confirm arbovirus infection in diagnostic laboratories and monitor immunological protection in vaccine recipients. Wild-type (wt) viruses used in the PRNT may be difficult to grow and plaque titrate, such as the dengue viruses (DENV), and/or may require biosafety level 3 (BSL3) containment, such as West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). These requirements preclude their use in diagnostic laboratories with only BSL2 capacity. In addition, wt JEV falls under the jurisdiction of the select-agent program and can be used only in approved laboratories. The chimeric vaccine viruses ChimeriVax-WNV and -SLEV have previously been shown to elicit antibody reactivity comparable to that of parental wt WNV and SLEV. ChimeriVax viruses provide advantages for PRNT, as follows: they grow more rapidly than most wt flaviviruses, produce large plaques, require BSL2 conditions, and are not under select-agent restrictions. We evaluated the ChimeriVax-DENV serotype 1 (DENV1), -DENV2, -DENV3, -DENV4, and -JEV for use in PRNT on sera from DENV- and JEV-infected patients and from JEV vaccine recipients. Serostatus agreement was 100% between the ChimeriVax-DENV serotypes and wt prototype DENV and 97% overall with ChimeriVax-JEV compared to prototype Nakayama JEV, 92% in a subgroup of JEV vaccine recipients, and 100% in serum from encephalitis patients naturally infected with JEV. ChimeriVax-DENV and -JEV plaque phenotype and BSL2 requirements, combined with sensitive and specific reactivity, make them good substitutes for wt DENV and JEV in PRNT in public health diagnostic laboratories.

Meteorological Conditions Associated with Increased Incidence of West Nile Virus Disease in the United States, 2004-2012

West Nile virus (WNV) is a leading cause of mosquito-borne disease in the United States. Annual seasonal outbreaks vary in size and location. Predicting where and when higher than normal WNV transmission will occur can help direct limited public health resources. We developed models for the contiguous United States to identify meteorological anomalies associated with above average incidence of WNV neuroinvasive disease from 2004 to 2012. We used county-level WNV data reported to ArboNET and meteorological data from the North American Land Data Assimilation System. As a result of geographic differences in WNV transmission, we divided the United States into East and West, and 10 climate regions. Above average annual temperature was associated with increased likelihood of higher than normal WNV disease incidence, nationally and in most regions. Lower than average annual total precipitation was associated with higher disease incidence in the eastern United States, but the opposite was true in most western regions. Although multiple factors influence WNV transmission, these findings show that anomalies in temperature and precipitation are associated with above average WNV disease incidence. Readily accessible meteorological data may be used to develop predictive models to forecast geographic areas with elevated WNV disease risk before the coming season.