Search

The case-fatality rate of yellow fever virus (YFV) is one of the highest among arthropod-borne viruses (arboviruses). Although historically, the Asia-Pacific region has remained free of YFV, the risk of introduction has never been higher due to the increasing influx of people from endemic regions and the recent outbreaks in Africa and South America. Singapore is a global hub for trade and tourism and therefore at high risk for YFV introduction. Effective control of the main domestic mosquito vector Aedes aegypti in Singapore has failed to prevent re-emergence of dengue, chikungunya and Zika viruses in the last two decades, raising suspicions that peridomestic mosquito species untargeted by domestic vector control measures may contribute to arbovirus transmission. Here, we provide empirical evidence that the peridomestic mosquito Aedes malayensis found in Singapore can transmit YFV. Our laboratory mosquito colony recently derived from wild Ae. malayensis in Singapore was experimentally competent for YFV to a similar level as Ae. aegypti controls. In addition, we captured Ae. malayensis females in one human-baited trap during three days of collection, providing preliminary evidence that host-vector contact may occur in field conditions. Finally, we detected Ae. malayensis eggs in traps deployed in high-rise building areas of Singapore. We conclude that Ae. malayensis is a competent vector of YFV and re-emphasize that vector control methods should be extended to target peridomestic vector species. [ABSTRACT FROM AUTHOR]

Introduction: The prevalence of bacteremia caused by Gram negative non-fermentative (GNNF) bacteria has been increasing globally over the past decade. Many studies have investigated their epidemiology but focus on the common GNNF including Pseudomonas aeruginosa and Acinetobacter baumannii. Knowledge of the uncommon GNNF bacteremias is very limited. This study explores invasive bloodstream infection GNNF isolates that were initially unidentified after testing with standard microbiological techniques. All isolations were made during laboratory-based surveillance activities in two rural provinces of Thailand between 2006 and 2014. Methods: A subset of GNNF clinical isolates (204/947), not identified by standard manual biochemical methodologies were run on the BD Phoenix automated identification and susceptibility testing system. If an organism was not identified (12/204) DNA was extracted for whole genome sequencing (WGS) on a MiSeq platform and data analysis performed using 3 web-based platforms: Taxonomer, CGE KmerFinder and One Codex. Results: The BD Phoenix automated identification system recognized 92% (187/204) of the GNNF isolates, and because of their taxonomic complexity and high phenotypic similarity 37% (69/187) were only identified to the genus level. Five isolates grew too slowly for identification. Antimicrobial sensitivity (AST) data was not obtained for 93/187 (50%) identified isolates either because of their slow growth or their taxa were not in the AST database associated with the instrument. WGS identified the 12 remaining unknowns, four to genus level only. Conclusion: The GNNF bacteria are of increasing concern in the clinical setting, and our inability to identify these organisms and determine their AST profiles will impede treatment. Databases for automated identification systems and sequencing annotation need to be improved so that opportunistic organisms are better covered. [ABSTRACT FROM AUTHOR]

Background: Lymphatic filariasis (LF) is a neglected tropical disease, and the Global Program to Eliminate LF delivers mass drug administration (MDA) to 500 million people every year. Adverse events (AEs) are common after LF treatment. Methodology/Principal findings: To better understand the pathogenesis of AEs, we studied LF-patients from a treatment trial. Plasma levels of many filarial antigens increased post-treatment in individuals with AEs, and this is consistent with parasite death. Circulating immune complexes were not elevated in these participants, and the classical complement cascade was not activated. Multiple cytokines increased after treatment in persons with AEs. A transcriptomic analysis was performed for nine individuals with moderate systemic AEs and nine matched controls. Differential gene expression analysis identified a significant transcriptional signature associated with post-treatment AEs; 744 genes were upregulated. The transcriptional signature was enriched for TLR and NF-κB signaling. Increased expression of seven out of the top eight genes upregulated in persons with AEs were validated by qRT-PCR, including TLR2. Conclusions/Significance: This is the first global study of changes in gene expression associated with AEs after treatment of lymphatic filariasis. Changes in cytokines were consistent with prior studies and with the RNAseq data. These results suggest that Wolbachia lipoprotein is involved in AE development, because it activates TLR2-TLR6 and downstream NF-κB. Additionally, LPS Binding Protein (LBP, which shuttles lipoproteins to TLR2) increased post-treatment in individuals with AEs. Improved understanding of the pathogenesis of AEs may lead to improved management, increased MDA compliance, and accelerated LF elimination. [ABSTRACT FROM AUTHOR]

Background: Human mobility plays a central role in shaping pathogen transmission by generating spatial and/or individual variability in potential pathogen-transmitting contacts. Recent research has shown that symptomatic infection can influence human mobility and pathogen transmission dynamics. Better understanding the complex relationship between symptom severity, infectiousness, and human mobility requires quantification of movement patterns throughout infectiousness. For dengue virus (DENV), human infectiousness peaks 0–2 days after symptom onset, making it paramount to understand human movement patterns from the beginning of illness. Methodology and principal findings: Through community-based febrile surveillance and RT-PCR assays, we identified a cohort of DENV+ residents of the city of Iquitos, Peru (n = 63). Using retrospective interviews, we measured the movements of these individuals when healthy and during each day of symptomatic illness. The most dramatic changes in mobility occurred during the first three days after symptom onset; individuals visited significantly fewer locations (Wilcoxon test, p = 0.017) and spent significantly more time at home (Wilcoxon test, p = 0.005), compared to when healthy. By 7–9 days after symptom onset, mobility measures had returned to healthy levels. Throughout an individual’s symptomatic period, the day of illness and their subjective sense of well-being were the most significant predictors for the number of locations and houses they visited. Conclusions/Significance: Our study is one of the first to collect and analyze human mobility data at a daily scale during symptomatic infection. Accounting for the observed changes in human mobility throughout illness will improve understanding of the impact of disease on DENV transmission dynamics and the interpretation of public health-based surveillance data. [ABSTRACT FROM AUTHOR]

Background: Zika virus (ZIKV) and Dengue virus (DENV) are often co-endemic. The high protein-sequence homology of flaviviruses renders IgG induced by and directed against them highly cross-reactive against their antigen(s), as observed on a large set of sera, leading to poorly reliable sero-diagnosis. Methods: We selected Domain III of the ZIKV Envelope (ZEDIII) sequence, which is virus specific. This recombinant domain was expressed and purified for the specific detection of ZEDIII-induced IgG by ELISA from ZIKV-RT-PCR-positive, ZIKV-IgM-positive, flavivirus-positive but ZIKV-negative, or flavivirus-negative sera. We also assessed the reactivity of ZEDIII-specific human antibodies against EDIII of DENV serotype 4 (D4EDIII) as a specific control. Sera from ZEDIII-immunized mice were also tested. Results: Cross-reactivity of IgG from 5,600 sera against total inactivated DENV or ZIKV was high (71.0% [69.1; 72.2]), whereas the specificity and sensitivity calculated using a representative cohort (242 sera) reached 90% [84.0; 95.8] and 92% [84.5; 99.5], respectively, using a ZEDIII-based ELISA. Moreover, purified human IgG against D2EDIII or D4EDIII did not bind to ZEDIII and we observed no D4EDIII reactivity with ZIKV-induced mouse polyclonal IgGs. Conclusions: We developed a ZEDIII-based ELISA that can discriminate between past or current DENV and ZIKV infections, allowing the detection of a serological scar from other flaviviruses. This could be used to confirm exposure of pregnant women or to follow the spread of an endemic disease. [ABSTRACT FROM AUTHOR]

Zika virus infection is associated with the development of Guillain-Barré syndrome (GBS), a neurological autoimmune disorder caused by immune recognition of gangliosides and other components at nerve membranes. Using a high-throughput ELISA, we have analyzed the anti-glycolipid antibody profile, including gangliosides, of plasma samples from patients with Zika infections associated or not with GBS in Salvador, Brazil. We have observed that Zika patients that develop GBS present higher levels of anti-ganglioside antibodies when compared to Zika patients without GBS. We also observed that a broad repertoire of gangliosides was targeted by both IgM and IgG anti-self antibodies in these patients. Since Zika virus infects neurons, which contain membrane gangliosides, antigen presentation of these infected cells may trigger the observed autoimmune anti-ganglioside antibodies suggesting direct infection-induced autoantibodies as a cause leading to GBS development. Collectively, our results establish a link between anti-ganglioside antibodies and Zika-associated GBS in patients. [ABSTRACT FROM AUTHOR]

Mother-to-child-transmission (MTCT) of human T-cell lymphotropic virus type-1(HTLV-1) contributes disproportionately to the burden of HTLV-1 associated diseases. All preventive measures to avoid MTCT rely on the identification of infected mothers. However, the impact of pregnancy on HTLV-1 diagnosis has not been clearly assessed. Paired samples from 21 HTLV-1 infected women taken during pregnancy and while not pregnant were analysed by CMIA and PCR. The signal-to-cut-off values (S/CO) were higher during pregnancy than in the paired non-pregnant samples. HTLV-1 proviral load did not alter significantly by pregnant state. S/CO positively correlated with HTLV proviral load. Pregnancy does not impair the diagnosis of HTLV-1/2, by either immunological (CMIA) or molecular (qPCR/nPCR) tests. [ABSTRACT FROM AUTHOR]

Background: Rift Valley fever (RVF) is one of the main vector borne zoonotic diseases that affects a wide range of ruminants and human beings in Africa and the Arabian Peninsula. A rapid and specific test for RVF diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures. Methodology/Principal findings: A first-line lateral flow immunochromatographic strip test (LFT) was developed for the detection of the nucleoprotein (N) of the RVF virus (RVFV). Its diagnostic performance characteristics were evaluated using reference stocks isolates recovered from different hosts and in geographic regions mimicking clinical specimens and from known RVF negative serum samples. A high level of diagnostic accuracy (DSe (35/35), DSp (167/169)) was observed, including the absence of cross-reactivity with viruses belonging to different genera. Conclusion/Significance: The fact no specialized reagents and laboratory equipment are needed, make this assay a valuable, first-line diagnostic tool in resource-poor diagnostic territories for on-site RVFV detection, however the staff require training. [ABSTRACT FROM AUTHOR]

Background: The diagnosis of schistosomiasis currently relies on microscopic detection of schistosome eggs in stool or urine samples and serological assays. The poor sensitivity of standard microscopic procedures performed in routine laboratories, makes molecular detection methods of increasing interest. The aim of the study was to evaluate two in-house real-time Schistosoma PCRs, targeting respectively S. mansoni [Sm] and S. haematobium [Sh] in excreta, biopsies and sera as potential tools to diagnose active infections and to monitor treatment efficacy. Methods: Schistosoma PCRs were performed on 412 samples (124 urine, 86 stools, 8 biopsies, 194 sera) from patients with suspected schistosomiasis, before anti-parasitic treatment. Results were compared to microscopic examination and serological assays (enzyme-linked immunosorbent assay (ELISA), indirect haemagglutination (HA) and Western Blot (WB) assay). Results: Compared to microscopy, PCRs significantly increased the sensitivity of diagnosis, from 4% to 10.5% and from 33.7% to 48.8%, for Sh in urine and Sm in stools, respectively. The overall sensitivity of PCR on serum samples was 72.7% and reached 94.1% in patients with positive excreta (microscopy). The specificity of serum PCR was 98.9%. After treatment, serum PCR positivity rates slowly declined from 93.8% at day 30 to 8% at day 360, whereas antibody detection remained positive after 1 year. Conclusion: Schistosoma PCRs clearly outperform standard microscopy on stools and urine and could be part of reference methods combined with WB-based serology, which remains a gold standard for initial diagnosis. When serological assays are positive and microscopy is negative, serum PCRs provide species information to guide further clinical exploration. Biomarkers such as DNA and antibodies are of limited relevance for early treatment monitoring but serum PCR could be useful when performed at least 1 year after treatment to help confirm a cured infection. [ABSTRACT FROM AUTHOR]

Background: Pathogens causing acute fever, with the exception of malaria, remain largely unidentified in sub-Saharan Africa, given the local unavailability of diagnostic tests and the broad differential diagnosis. Methodology: We conducted a cross-sectional study including outpatient acute undifferentiated fever in both children and adults, between November 2015 and June 2016 in Kinshasa, Democratic Republic of Congo. Serological and molecular diagnostic tests for selected arboviral infections were performed on blood, including PCR, NS1-RDT, ELISA and IFA for acute, and ELISA and IFA for past infections. Results: Investigation among 342 patients, aged 2 to 68 years (mean age of 21 years), with acute undifferentiated fever (having no clear focus of infection) revealed 19 (8.1%) acute dengue–caused by DENV-1 and/or DENV-2 –and 2 (0.9%) acute chikungunya infections. Furthermore, 30.2% and 26.4% of participants had been infected in the past with dengue and chikungunya, respectively. We found no evidence of acute Zika nor yellow fever virus infections. 45.3% of patients tested positive on malaria Rapid Diagnostic Test, 87.7% received antimalarial treatment and 64.3% received antibacterial treatment. Discussion: Chikungunya outbreaks have been reported in the study area in the past, so the high seroprevalence is not surprising. However, scarce evidence exists on dengue transmission in Kinshasa and based on our data, circulation is more important than previously reported. Furthermore, our study shows that the prescription of antibiotics, both antibacterial and antimalarial drugs, is rampant. Studies like this one, elucidating the causes of acute fever, may lead to a more considerate and rigorous use of antibiotics. This will not only stem the ever-increasing problem of antimicrobial resistance, but will–ultimately and hopefully–improve the clinical care of outpatients in low-resource settings. Trial registration: ClinicalTrials.gov . [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a newly-identified infectious cause of congenital disease. Transplacental transfer of maternal IgG to the fetus plays an important role in preventing many neonatal infections. However, antibody transfer may also have negative consequences, such as mediating enhancement of flavivirus infections in early life, or trafficking of virus immune complexes to the fetal compartment. ZIKV infection produces placental pathology which could lead to impaired IgG transfer efficiency as occurs in other maternal infections, such as HIV-1 and malaria. In this study, we asked whether ZIKV infection during pregnancy impairs transplacental transfer of IgG. We enrolled pregnant women with fever or rash in a prospective cohort in Vitoria, Brazil during the recent ZIKV epidemic. ZIKV and dengue virus (DENV)-specific IgG, ZIKV and DENV neutralizing antibodies, and routine vaccine antigen-specific IgG were measured in maternal samples collected around delivery and 20 paired cord blood samples. We concluded that 8 of these mothers were infected with ZIKV during pregnancy and 12 were ZIKV-uninfected. The magnitude of flavivirus-specific IgG, neutralizing antibody, and vaccine-elicited IgG were highly correlated between maternal plasma and infant cord blood in both ZIKV-infected and -uninfected mother-infant pairs. Moreover, there was no difference in the magnitude of plasma flavivirus-specific IgG levels between mothers and infants regardless of ZIKV infection status. Our data suggests that maternal ZIKV infection during pregnancy does not impair the efficiency of placental transfer of flavivirus-specific, functional, and vaccine-elicited IgG. These findings have implications for the neonatal outomes of maternal ZIKV infection and optimal administration of antibody-based ZIKV vaccines and therapeutics. [ABSTRACT FROM AUTHOR]

Background: We investigated the relationship between bacterial load in Buruli ulcer (BU) lesions and the development of paradoxical reaction following initiation of antibiotic treatment. Methods: This was a longitudinal study involving BU patients from June 2013 to June 2017. Fine needle aspirates (FNA) and swab samples were obtained to establish the diagnosis of BU by PCR. Additional samples were obtained at baseline, during and after treatment (if the lesion had not healed) for microscopy, culture and combined 16S rRNA reverse transcriptase/ IS2404 qPCR assay. Patients were followed up at regular intervals until complete healing. Results: Forty-seven of 354 patients (13%) with PCR confirmed BU had a PR, occurring between 2 and 42 (median 6) weeks after treatment initiation. The bacterial load, the proportion of patients with positive M. ulcerans culture (15/34 (44%) vs 29/119 (24%), p = 0.025) and the proportion with positive microscopy results (19/31 (61%) vs 28/90 (31%), p = 0.003) before initiation of treatment were significantly higher in the PR compared to the no PR group. Plaques (OR 5.12; 95% CI 2.26–11.61; p<0.001), oedematous (OR 4.23; 95% CI 1.43–12.5; p = 0.009) and category II lesions (OR 2.26; 95% CI 1.14–4.48; p = 0.02) were strongly associated with the occurrence of PR. The median time to complete healing (28 vs 13 weeks, p <0.001) was significantly longer in the PR group. Conclusions: Buruli ulcer patients who develop PR are characterized by high bacterial load in lesion samples taken at baseline and a higher rate of positive M. ulcerans culture. Occurrence of a PR was associated with delayed healing. Trial registration: ClinicalTrials.gov . [ABSTRACT FROM AUTHOR]

Background: Histoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly due to delayed diagnosis, caused by slow fungal growth in culture. Therefore, a fast, suitable and cost-effective assay is required for the diagnosis of histoplasmosis in resource-limited laboratories. This study aimed to develop and evaluate two new molecular approaches for a more cost-effective diagnosis of histoplasmosis. Methodology: Seeking a fast, suitable, sensitive, specific and low-cost molecular detection technique, we developed a new Loop-mediated Isothermal Amplification (LAMP) assay and nested PCR, both targeting the Internal Transcribed Spacer (ITS) multicopy region of Histoplasma capsulatum. The sensitivity was evaluated using 26 bone marrow and 1 whole blood specimens from patients suspected to have histoplasmosis and 5 whole blood samples from healthy subjects. All specimens were evaluated in culture, as a reference standard test, and Hcp100 nPCR, as a molecular reference test. A heparin-containing whole blood sample from a heathy subject was spiked with H. capsulatum cells and directly assayed with no previous DNA extraction. Results: Both assays were able to detect down to 1 fg/μL of H. capsulatum DNA, and ITS LAMP results could also be revealed to the naked-eye by adding SYBR green to the reaction tube. In addition, both assays were able to detect all clades of Histoplasma capsulatum cryptic species complex. No cross-reaction with other fungal pathogens was presented. In comparison with Hcp100 nPCR, both assays reached 83% sensitivity and 92% specificity. Furthermore, ITS LAMP assay showed no need for DNA extraction, since it could be directly applied to crude whole blood specimens, with a limit of detection of 10 yeasts/μL. Conclusion: ITS LAMP and nPCR assays have the potential to be used in conjunction with culture for early diagnosis of progressive disseminated histoplasmosis, allowing earlier, appropriate treatment of the patient. The possibility of applying ITS LAMP, as a direct assay, with no DNA extraction and purification steps, makes it suitable for resource-limited laboratories. However, more studies are necessary to validate ITS LAMP and nPCR as direct assay in other types of clinical specimens. [ABSTRACT FROM AUTHOR]

Ticks successfully feed and transmit pathogens by injecting pharmacological compounds in saliva to thwart host defenses. We have previously used LC-MS/MS to identify proteins that are present in saliva of unfed Amblyomma americanum ticks that were exposed to different hosts. Here we show that A. americanum serine protease inhibitor (serpin) 27 (AAS27) is an immunogenic saliva protein that is injected into the host within the first day of tick feeding and is an anti-inflammatory protein that might act by blocking plasmin and trypsin functions. Although AAS27 is injected into the host throughout tick feeding, qRT-PCR and western blotting analyses indicate that the respective transcript and protein are present in high amounts within the first 24 h of tick feeding. Biochemical screening of Pichia pastoris-expressed recombinant (r) AAS27 against mammalian proteases related to host defense shows it is an inhibitor of trypsin and plasmin, with stoichiometry of inhibition indices of 3.5 and 3.8, respectively. Consistent with typical inhibitory serpins, rAAS27 formed heat- and SDS-stable irreversible complexes with both proteases. We further demonstrate that rAAS27 inhibits trypsin with ka of 6.46 ± 1.24 x 104 M-1 s-1, comparable to serpins of other tick species. We show that native AAS27 is part of the repertoire of proteins responsible for the inhibitory activity against trypsin in crude tick saliva. AAS27 is likely utilized by the tick to evade the hosts inflammation defense since rAAS27 blocks both formalin and compound 48/80-induced inflammation in rats. Tick immune sera of rabbits that had acquired resistance against tick feeding following repeated infestations with A. americanum or Ixodes scapularis ticks reacts with rAAS27. Of significant interest, antibody to rAAS27 blocks this serpin inhibitory functions. Taken together, we conclude that AAS27 is an anti-inflammatory protein secreted into the host during feeding and may represent a potential candidate for development of an anti-tick vaccine. [ABSTRACT FROM AUTHOR]

The explosive spread of Zika virus (ZIKV) and associated complications in flavivirus-endemic regions underscore the need for sensitive and specific serodiagnostic tests to distinguish ZIKV, dengue virus (DENV) and other flavivirus infections. Compared with traditional envelope protein-based assays, several nonstructural protein 1 (NS1)-based assays showed improved specificity, however, none can detect and discriminate three flaviviruses in a single assay. Moreover, secondary DENV infection and ZIKV infection with previous DENV infection, both common in endemic regions, cannot be discriminated. In this study, we developed a high-throughput and multiplex IgG microsphere immunoassay (MIA) using the NS1 proteins of DENV1-DENV4, ZIKV and West Nile virus (WNV) to test samples from reverse-transcription-polymerase-chain reaction-confirmed cases, including primary DENV1, DENV2, DENV3, WNV and ZIKV infections, secondary DENV infection, and ZIKV infection with previous DENV infection. Combination of four DENV NS1 IgG MIAs revealed a sensitivity of 94.3% and specificity of 97.2% to detect DENV infection. The ZIKV and WNV NS1 IgG MIAs had a sensitivity/specificity of 100%/87.9% and 86.1%/78.4%, respectively. A positive correlation was found between the readouts of enzyme-linked immunosorbent assay and MIA for different NS1 tested. Based on the ratio of relative median fluorescence intensity of ZIKV NS1 to DENV1 NS1, the IgG MIA can distinguish ZIKV infection with previous DENV infection and secondary DENV infection with a sensitivity of 88.9–90.0% and specificity of 91.7–100.0%. The multiplex and high-throughput assay could be applied to serodiagnosis and serosurveillance of DENV, ZIKV and WNV infections in endemic regions. [ABSTRACT FROM AUTHOR]

Urbanization is one of the major drivers of dengue epidemics globally. In Kenya, an intriguing pattern of urban dengue virus epidemics has been documented in which recurrent epidemics are reported from the coastal city of Mombasa, whereas no outbreaks occur in the two major inland cities of Kisumu and Nairobi. In an attempt to understand the entomological risk factors underlying the observed urban dengue epidemic pattern in Kenya, we evaluated vector density, human feeding patterns, vector genetics, and prevailing environmental temperature to establish how these may interact with one another to shape the disease transmission pattern. We determined that (i) Nairobi and Kisumu had lower vector density and human blood indices, respectively, than Mombasa, (ii) vector competence for dengue-2 virus was comparable among Ae. aegypti populations from the three cities, with no discernible association between susceptibility and vector cytochrome c oxidase subunit 1 gene variation, and (iii) vector competence was temperature-dependent. Our study suggests that lower temperature and Ae. aegypti vector density in Nairobi may be responsible for the absence of dengue outbreaks in the capital city, whereas differences in feeding behavior, but not vector competence, temperature, or vector density, contribute in part to the observed recurrent dengue epidemics in coastal Mombasa compared to Kisumu. [ABSTRACT FROM AUTHOR]

Bacillus anthracis and Yersinia pestis are zoonotic bacteria capable of causing severe and sometimes fatal infections in animals and humans. Although considered as diseases of antiquity in industrialized countries due to animal and public health improvements, they remain endemic in vast regions of the world disproportionally affecting the poor. These pathogens also remain a serious threat if deployed in biological warfare. A single vaccine capable of stimulating rapid protection against both pathogens would be an extremely advantageous public health tool. We produced multiple-antigen fusion proteins (MaF1 and MaF2) containing protective regions from B. anthracis protective antigen (PA) and lethal factor (LF), and from Y. pestis V antigen (LcrV) and fraction 1 (F1) capsule. The MaF2 sequence was also expressed from a plasmid construct (pDNA-MaF2). Immunogenicity and protective efficacy were investigated in mice following homologous and heterologous prime-boost immunization. Antibody responses were determined by ELISA and anthrax toxin neutralization assay. Vaccine efficacy was determined against lethal challenge with either anthrax toxin or Y. pestis. Both constructs elicited LcrV and LF-specific serum IgG, and MaF2 elicited toxin-neutralizing antibodies. Immunizations with MaF2 conferred 100% and 88% protection against Y. pestis and anthrax toxin, respectively. In contrast, pDNA-MaF2 conferred only 63% protection against Y. pestis and no protection against anthrax toxin challenge. pDNA-MaF2-prime MaF2-boost induced 75% protection against Y. pestis and 25% protection against anthrax toxin. Protection was increased by the molecular adjuvant CARDif. In conclusion, MaF2 is a promising multi-antigen vaccine candidate against anthrax and plague that warrants further investigation. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV), an emerging arbovirus belonging to the genus Flavivirus, is transmitted by Aedes mosquitoes. ZIKV infection can cause microcephaly of newborn babies and Guillain–Barré syndrome in adults. Because no licensed vaccine or specific antiviral treatment is available for ZIKV infection, the most commonly used approach to control the spread of ZIKV is suppression of the mosquito vector population. A novel proposed strategy to block arthropod virus (arbovirus) transmission is based on the chemical inhibition of virus infection in mosquitoes. However, only a few drugs and compounds have been tested with such properties. Here we present a comprehensive screen of 55 FDA-approved anti-flaviviral drugs for potential anti-ZIKV and mosquitocidal activity. Four drugs (auranofin, actinomycin D (Act-D), bortezomib and gemcitabine) were toxic to C6/36 cells, and two drugs (5-fluorouracil and mycophenolic acid (MPA)) significantly reduced ZIKV production in C6/36 cells at 2 μM and 0.5 μM, respectively. Three drugs (Act-D, cyclosporin A, ivermectin) exhibited a strong adulticidal activity, and six drugs (U18666A, retinoic acid p-hydroxyanilide (4-HPR), clotrimazole, bortezomib, MPA, imatinib mesylate) significantly suppressed ZIKV infection in mosquito midguts. Some of these FDA-approved drugs may have potential for use for the development of ZIKV transmission-blocking strategies. [ABSTRACT FROM AUTHOR]

Background: To be transmitted to vertebrate hosts via the saliva of their vectors, arthropod-borne viruses have to cross several barriers in the mosquito body, including the midgut infection and escape barriers. Yellow fever virus (YFV) belongs to the genus Flavivirus, which includes human viruses transmitted by Aedes mosquitoes, such as dengue and Zika viruses. The live-attenuated YFV-17D vaccine has been used safely and efficiently on a large scale since the end of World War II. Early studies have shown, using viral titration from salivary glands of infected mosquitoes, that YFV-17D can infect Aedes aegypti midgut, but does not disseminate to other tissues. Methodology/Principal findings: Here, we re-visited this issue using a panel of techniques, such as RT-qPCR, Western blot, immunofluorescence and titration assays. We showed that YFV-17D replication was not efficient in Aedes aegypti midgut, as compared to the clinical isolate YFV-Dakar. Viruses that replicated in the midgut failed to disseminate to secondary organs. When injected into the thorax of mosquitoes, viruses succeeded in replicating into midgut-associated tissues, suggesting that, during natural infection, the block for YFV-17D replication occurs at the basal membrane of the midgut. Conclusions/Significance: The two barriers associated with Ae. aegypti midgut prevent YFV-17D replication. Our study contributes to our basic understanding of vector–pathogen interactions and may also aid in the development of non-transmissible live virus vaccines. [ABSTRACT FROM AUTHOR]

Enteric fevers, caused by the Salmonella enterica serovars Typhi (ST), Paratyphi A (PA) and Paratyphi B (PB), are life-threatening illnesses exhibiting very similar clinical symptoms but with distinct epidemiologies, geographical distributions and susceptibilities to antimicrobial treatment. Nevertheless, the mechanisms by which the host recognizes pathogens with high levels of homology, such as these bacterial serovars, remain poorly understood. Using a three-dimensional organotypic model of the human intestinal mucosa and PA, PB, and ST, we observed significant differences in the secretion patterns of pro-inflammatory cytokines and chemokines elicited by these serovars. These cytokines/chemokines were likely to be co-regulated and influenced the function of epithelial cells, such as the production of IL-8. We also found differing levels of polymorphonuclear leukocyte (PMN) migration among various infection conditions that either included or excluded lymphocytes and macrophages (Mϕ), strongly suggesting feedback mechanisms among these cells. Blocking experiments showed that IL-1β, IL-6, IL-8, TNF-α and CCL3 cytokines were involved in the differential regulation of migration patterns. We conclude that the crosstalk among the lymphocytes, Mϕ, PMN and epithelial cells is cytokine/chemokine-dependent and bacterial-serotype specific, and plays a pivotal role in orchestrating the functional efficiency of the innate cells and migratory characteristics of the leukocytes. [ABSTRACT FROM AUTHOR]

The 2013–2016 Ebola virus outbreak in West Africa was the largest and deadliest outbreak to date. Here we conducted a serological study to examine the antibody levels in survivors and the seroconversion in close contacts who took care of Ebola-infected individuals, but did not develop symptoms of Ebola virus disease. In March 2017, we collected blood samples from 481 individuals in Makeni, Sierra Leone: 214 survivors and 267 close contacts. Using commercial, quantitative ELISAs, we tested the plasma for IgG-specific antibodies against three major viral antigens: GP, the only viral glycoprotein expressed on the virus surface; NP, the most abundant viral protein; and VP40, a major structural protein of Zaire ebolavirus. We also determined neutralizing antibody titers. In the cohort of Ebola survivors, 97.7% of samples (209/214) had measurable antibody levels against GP, NP, and/or VP40. Of these positive samples, all but one had measurable neutralizing antibody titers against Ebola virus. For the close contacts, up to 12.7% (34/267) may have experienced a subclinical virus infection as indicated by detectable antibodies against GP. Further investigation is warranted to determine whether these close contacts truly experienced subclinical infections and whether these asymptomatic infections played a role in the dynamics of transmission. [ABSTRACT FROM AUTHOR]

Background: Oral cholera vaccine (OCV) containing killed Vibrio cholerae O1 and O139 organisms (Bivalent-OCV; Biv-OCV) are playing a central role in global cholera control strategies. OCV is currently administered in a 2-dose regimen (day 0 and 14). There is a growing body of evidence that immune responses targeting the O-specific polysaccharide (OSP) of V. cholerae mediate protection against cholera. There are limited data on anti-OSP responses in recipients of Biv-OCV. We assessed serum antibody responses against O1 OSP, as well as antibody secreting cell (ASC) responses (a surrogate marker for mucosal immunity) and memory B cell responses in blood of adult recipients of Biv-OCV in Dhaka, Bangladesh. Methodology/Principal findings: We enrolled 30 healthy adults in this study and administered two doses of OCV (Shanchol) at days 0 and 14. Blood samples were collected before vaccination (day 0) and 7 days after each vaccination (day 7 and day 21), as well as on day 44. Serum responses were largely IgA with minimal IgG and IgM responses in this population. There was no appreciable boosting following day 14 vaccination. There were significant anti-OSP IgA ASC responses on day 7 following the first vaccination, but none after the second immunization. Anti-OSP IgA memory B cell responses were detectable 30 days after completion of the vaccination series, with no evident induction of IgG memory responses. In this population, anti-Ogawa OSP responses were more prominent than anti-Inaba responses, perhaps reflecting impact of previous exposure. Serum anti-OSP responses returned to baseline within 30 days of completing the vaccine series. Conclusion: Our results call into question the utility of the 2-dose regimen separated by 14 days in adults in cholera endemic areas, and also suggest that Biv-OCV-induced immune responses targeting OSP are largely IgA in this highly endemic cholera area. Studies in children in cholera-endemic areas need to be performed. Protective efficacy that extends for more than a month after vaccination presumably is mediated by direct mucosal immune response which is not assessed in this study. Our results suggest a single dose of OCV in adults in a cholera endemic zone may be sufficient to mediate at least short-term protection. [ABSTRACT FROM AUTHOR]

Viroporins like influenza A virus M2, hepatitis C virus p7, HIV-1 Vpu and picornavirus 2B associate with host membranes, and create hydrophilic corridors, which are critical for viral entry, replication and egress. The 6K proteins from alphaviruses are conjectured to be viroporins, essential during egress of progeny viruses from host membranes, although the analogue in Chikungunya Virus (CHIKV) remains relatively uncharacterized. Using a combination of electrophysiology, confocal and electron microscopy, and molecular dynamics simulations we show for the first time that CHIKV 6K is an ion channel forming protein that primarily associates with endoplasmic reticulum (ER) membranes. The ion channel activity of 6K can be inhibited by amantadine, an antiviral developed against the M2 protein of Influenza A virus; and CHIKV infection of cultured cells can be effectively inhibited in presence of this drug. Our study provides crucial mechanistic insights into the functionality of 6K during CHIKV-host interaction and suggests that 6K is a potential therapeutic drug target, with amantadine and its derivatives being strong candidates for further development. [ABSTRACT FROM AUTHOR]

Typhoid fever is endemic across sub-Saharan Africa. However, estimates of the burden of typhoid are undermined by insufficient blood volumes and lack of sensitivity of blood culture. Here, we aimed to address this limitation by exploiting pre-enrichment culture followed by PCR, alongside routine blood culture to improve typhoid case detection. We carried out a prospective diagnostic cohort study and enrolled children (aged 0–4 years) with non-specific febrile disease admitted to a tertiary hospital in Blantyre, Malawi from August 2014 to July 2016. Blood was collected for culture (BC) and real-time PCR after a pre-enrichment culture in tryptone soy broth and ox-bile. DNA was subjected to PCR for invA (Pan-Salmonella), staG (S. Typhi), and fliC (S. Typhimurium) genes. A positive PCR was defined as invA plus either staG or fliC (CT<29). IgM and IgG ELISA against four S. Typhi antigens was also performed. In total, 643 children (median age 1.3 years) with nonspecific febrile disease were enrolled; 31 (4.8%) were BC positive for Salmonella (n = 13 S. Typhi, n = 16 S. Typhimurium, and n = 2 S. Enteritidis). Pre-enrichment culture of blood followed by PCR identified a further 8 S. Typhi and 15 S. Typhimurium positive children. IgM and IgG titres to the S. Typhi antigen STY1498 (haemolysin) were significantly higher in children that were PCR positive but blood culture negative compared to febrile children with all other non-typhoid illnesses. The addition of pre-enrichment culture and PCR increased the case ascertainment of invasive Salmonella disease in children by 62–94%. These data support recent burden estimates that highlight the insensitivity of blood cultures and support the targeting of pre-school children for typhoid vaccine prevention in Africa. Blood culture with real-time PCR following pre-enrichment should be used to further refine estimates of vaccine effectiveness in typhoid vaccine trials. [ABSTRACT FROM AUTHOR]

The N-linked glycosylation motif at amino acid position 154–156 of the envelope (E) protein of West Nile virus (WNV) is linked to enhanced murine neuroinvasiveness, avian pathogenicity and vector competence. Naturally occurring isolates with altered E protein glycosylation patterns have been observed in WNV isolates; however, the specific effects of these polymorphisms on avian host pathogenesis and vector competence have not been investigated before. In the present study, amino acid polymorphisms, NYT, NYP, NYF, SYP, SYS, KYS and deletion (A’DEL), were reverse engineered into a parental WNV (NYS) cDNA infectious clone to generate WNV glycosylation mutant viruses. These WNV glycosylation mutant viruses were characterized for in vitro growth, pH-sensitivity, temperature-sensitivity and host competence in American crows (AMCR), house sparrows (HOSP) and Culex quinquefasciatus. The NYS and NYT glycosylated viruses showed higher viral replication, pH and temperature sensitivity than NYP, NYF, SYP, SYS, KYS and A’DEL viruses in vitro. Interestingly, in vivo results demonstrated asymmetric effects in avian and mosquito competence that were independent of the E-protein glycosylation status. In AMCRs and HOSPs, all viruses showed comparable viremias with the exception of NYP and KYS viruses that showed attenuated phenotypes. Only NYP showed reduced vector competence in both Cx. quinquefasciatus and Cx. tarsalis. Glycosylated NYT exhibited similar avian virulence properties as NYS, but resulted in higher mosquito oral infectivity than glycosylated NYS and nonglycosylated, NYP, NYF, SYP and KYS mutants. These data demonstrated that amino acid polymorphisms at E154/156 dictate differential avian host and vector competence phenotypes independent of E-protein glycosylation status. [ABSTRACT FROM AUTHOR]

Background: Glanders caused by Burkholderia mallei is a re-emerging zoonotic disease affecting solipeds and humans. Furthermore, B. mallei is genetically related to B. pseudomallei, which is the causative agent of melioidosis. Both facultative intracellular bacteria are classified as tier 1 select biothreat agents. Our previous study with a B. mallei ΔtonB Δhcp1 (CLH001) live-attenuated vaccine demonstrated that it is attenuated, safe and protective against B. mallei wild-type strain in the susceptible BALB/c mouse model. Methodology/Principal finding: In our current work, we evaluated the protective efficacy of CLH001 against glanders and melioidosis in the more disease-resistant C57BL/6 mouse strain. The humoral as well as cellular immune responses were also examined. We found that CLH001-immunized mice showed 100% survival against intranasal and aerosol challenge with B. mallei ATCC 23344. Moreover, this vaccine also afforded significant cross-protection against B. pseudomallei K96243, with low level bacterial burden detected in organs. Immunization with a prime and boost regimen of CLH001 induced significantly greater levels of total and subclasses of IgG, and generated antigen-specific splenocyte production of IFN-γ and IL-17A. Interestingly, protection induced by CLH001 is primarily dependent on humoral immunity, while CD4+ and CD8+ T cells played a less critical protective role. Conclusions/Significance: Our data indicate that CLH001 serves as an effective live attenuated vaccine to prevent glanders and melioidosis. The quantity and quality of antibody responses as well as improving cell-mediated immune responses following vaccination need to be further investigated prior to advancement to preclinical studies. [ABSTRACT FROM AUTHOR]

The prevalence of rheumatic heart disease (RHD) in the Aboriginal population of the Australian Northern Territory is high, and Streptococcus pyogenes skin infections likely contribute to this. A promising candidate S. pyogenes “30mer” vaccine is composed of 30 pharyngitis associated type-specific antigens from the S. pyogenes M protein. Cross opsonisation experiments suggest that 30mer vaccine protection may extend to non-cognate emm types. A new “emm cluster” scheme for classifying M protein is based on the full-length coding sequence, and correlates with functional and immunological properties, and anatomical tropism. Twenty-seven years of research in the Northern Territory has yielded 1810 S. pyogenes isolates with clinical and emm type data. The primary aim was to analyse these data with reference to the emm cluster scheme and cross opsonisation information, to inform estimation of 30mer vaccine efficacy in the Northern Territory. The isolates encompass 101 emm types. Variants of cluster A-C were enriched in throat isolates, and variants of emm cluster D enriched in skin isolates. Throat isolates were enriched for 30mer vaccine cognate emm types in comparison with skin isolates of which only 25% were vaccine emm types. While cross opsonisation data indicates potential for enhancing 30mer vaccine coverage, more than one third of skin isolates were within 38 emm types untested for cross opsonisation. Emm cluster D variants, in particular emm cluster D4, were not only all non-cognate with the vaccine, but were abundant and diverse, and less likely to be cross-opsonisation positive than other emm clusters. Long term persistence of many emm types in the study area was revealed. It was concluded that the 30mer vaccine efficacy in the Northern Territory will likely require both cross protection, and additional measures to elicit immunity against variants of emm cluster D. [ABSTRACT FROM AUTHOR]

Giardia lamblia is a common intestinal parasitic infection that although often acutely asymptomatic, is associated with debilitating chronic intestinal and extra-intestinal sequelae. In previously healthy adults, a primary sporadic Giardia infection can lead to gut dysfunction and fatigue. These symptoms correlate with markers of inflammation that persist well after the infection is cleared. In contrast, in endemic settings, first exposure occurs in children who are frequently malnourished and also co-infected with other enteropathogens. In these children, Giardia rarely causes symptoms and associates with several decreased markers of inflammation. Mechanisms underlying these disparate and potentially enduring outcomes following Giardia infection are not presently well understood. A body of work suggests that the outcome of experimental Giardia infection is influenced by the nutritional status of the host. Here, we explore the consequences of experimental Giardia infection under conditions of protein sufficiency or deficiency on cytokine responses of ex vivo bone marrow derived dendritic cells (BMDCs) to endotoxin stimulation. We show that BMDCs from Giardia- challenged mice on a protein sufficient diet produce more IL-23 when compared to uninfected controls whereas BMDCs from Giardia challenged mice fed a protein deficient diet do not. Further, in vivo co-infection with Giardia attenuates robust IL-23 responses in endotoxin-stimulated BMDCs from protein deficient mice harboring enteroaggregative Escherichia coli. These results suggest that intestinal Giardia infection may have extra-intestinal effects on BMDC inflammatory cytokine production in a diet dependent manner, and that Giardia may influence the severity of the innate immune response to other enteropathogens. This work supports recent findings that intestinal microbial exposure may have lasting influences on systemic inflammatory responses, and may provide better understanding of potential mechanisms of post-infectious sequelae and clinical variation during Giardia and enteropathogen co-infection. [ABSTRACT FROM AUTHOR]

Background: Acute kidney injury (AKI) is a major complication of snake envenoming, but early diagnosis remains problematic. We aimed to investigate the time course of novel renal biomarkers in AKI following Russell’s viper (Daboia russelii) bites. Methodology/Principal findings: We recruited a cohort of patients with definite Russell’s viper envenoming and collected serial blood and urine samples on admission (<4h post-bite), 4-8h, 8-16h, 16-24h, 1 month and 3 months post-bite. AKI stage (1–3) was defined using the Acute Kidney Injury Network criteria. AKI stages (1–3) were defined by the Acute Kidney Injury Network (AKIN) criteria. There were 65 Russell’s viper envenomings and 49 developed AKI: 24 AKIN stage 1, 13 stage 2 and 12 stage 3. There was a significant correlation between venom concentrations and AKI stage (p = 0.007), and between AKI stage and six peak biomarker concentrations. Although most biomarker concentrations were elevated within 8h, no biomarker performed well in diagnosing AKI <4h post-bite. Three biomarkers were superior to serum creatinine (sCr) in predicting AKI (stage 2/3) 4-8h post-bite: serum cystatin C (sCysC) with an area under the receiver operating curve (AUC-ROC), 0.78 (95%CI:0.64–0.93), urine neutrophil gelatinase-associated lipocalin (uNGAL), 0.74 (95%CI:0.59–0.87) and urine clusterin (uClu), 0.81 (95%CI:0.69–0.93). No biomarker was better than sCr after 8h. Six other urine biomarkers urine albumin, urine beta2-microglobulin, urine kidney injury molecule-1, urine cystatin C, urine trefoil factor-3 and urine osteopontin either had minimal elevation, and/or minimal prediction for AKI stage 2/3 (AUC-ROC<0.7). Conclusions/Significance: AKI was common and sometimes severe following Russell’s viper bites. Three biomarkers uClu, uNGAL and sCysC, appeared to become abnormal in AKI earlier than sCr, and may be useful in early identification of envenoming. [ABSTRACT FROM AUTHOR]

Background: T cell immunoglobulin mucin domain-1 (TIM-1) is a phosphatidylserine (PS) receptor, mediating filovirus entry into cells through interactions with PS on virions. TIM-1 expression has been implicated in Ebola virus (EBOV) pathogenesis; however, it remains unclear whether this is due to TIM-1 serving as a filovirus receptor in vivo or, as others have suggested, TIM-1 induces a cytokine storm elicited by T cell/virion interactions. Here, we use a BSL2 model virus that expresses EBOV glycoprotein to demonstrate the importance of TIM-1 as a virus receptor late during in vivo infection. Methodology/Principal findings: Infectious, GFP-expressing recombinant vesicular stomatitis virus encoding either full length EBOV glycoprotein (EBOV GP/rVSV) or mucin domain deleted EBOV glycoprotein (EBOV GPΔO/rVSV) was used to assess the role of TIM-1 during in vivo infection. GFP-expressing rVSV encoding its native glycoprotein G (G/rVSV) served as a control. TIM-1-sufficient or TIM-1-deficient BALB/c interferon α/β receptor-/- mice were challenged with these viruses. While G/rVSV caused profound morbidity and mortality in both mouse strains, TIM-1-deficient mice had significantly better survival than TIM-1-expressing mice following EBOV GP/rVSV or EBOV GPΔO/rVSV challenge. EBOV GP/rVSV or EBOV GPΔO/rVSV in spleen of infected animals was high and unaffected by expression of TIM-1. However, infectious virus in serum, liver, kidney and adrenal gland was reduced late in infection in the TIM-1-deficient mice, suggesting that virus entry via this receptor contributes to virus load. Consistent with higher virus loads, proinflammatory chemokines trended higher in organs from infected TIM-1-sufficient mice compared to the TIM-1-deficient mice, but proinflammatory cytokines were more modestly affected. To assess the role of T cells in EBOV GP/rVSV pathogenesis, T cells were depleted in TIM-1-sufficient and -deficient mice and the mice were challenged with virus. Depletion of T cells did not alter the pathogenic consequences of virus infection. Conclusions: Our studies provide evidence that at late times during EBOV GP/rVSV infection, TIM-1 increased virus load and associated mortality, consistent with an important role of this receptor in virus entry. This work suggests that inhibitors which block TIM-1/virus interaction may serve as effective antivirals, reducing virus load at late times during EBOV infection. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax and Hepatitis B virus (HBV) are globally outspread in similar geographic regions. The concurrence of both infections and its association with some degree of protection against symptomatic and/or severe vivax malaria has been already described. Nevertheless, data on how host response to both pathogens undermines the natural progression of the malarial infection are scarce. Here, a large cohort of vivax malaria and HBV patients is retrospectively analyzed in an attempt to depict how inflammatory characteristics could be potentially related to the protection to severe malaria in coinfection. Methods: A retrospective analysis of a databank containing 601 individuals from the Brazilian Amazon, including 179 symptomatic P. vivax monoinfected patients, 145 individuals with asymptomatic P. vivax monoinfection, 28 P. vivax-HBV coinfected patients, 29 HBV monoinfected subjects and 165 healthy controls, was performed. Data on plasma levels of multiple chemokines, cytokines, acute phase proteins, hepatic enzymes, bilirubin and creatinine were analyzed to describe and compare biochemical profiles associated to each type of infection. Results: Coinfected individuals predominantly presented asymptomatic malaria, referred increased number of previous malaria episodes than symptomatic vivax-monoinfected patients, and were predominantly younger than asymptomatic vivax-monoinfected individuals. Coinfection was hallmarked by substantially elevated concentrations of interleukin (IL)-10 and heightened levels of C-C motif chemokine ligand (CCL)2. Correlation matrices showed that coinfected individuals presented a distinct biomarker profile when compared to asymptomatic or symptomatic P. vivax patients, or HBV-monoinfected individuals. Parasitemia could distinguish coinfected from symptomatic or asymptomatic P. vivax-monoinfected patients. HBV viremia was associated to distinct inflammatory profiles in HBV-monoinfected and coinfected patients. Conclusion: The findings demonstrate a distinct inflammatory profile in coinfected patients, with characteristics associated with immune responses to both pathogens. These host responses to P. vivax and HBV, in conjunction, could then be potentially associated, if not mainly responsible, for the protection against symptomatic vivax malaria. [ABSTRACT FROM AUTHOR]

The definitive method for diagnosis of porcine cysticercosis is the detection of cysticerci at necropsy. Cysts are typically located in the striated muscle and brain. Until recently Taenia solium cysticerci have not been definitively identified in other tissue locations, despite several comprehensive investigations having been undertaken which included investigation of body organs other than muscle and brain. Recently a study conducted in Zambia reported 27% infection with T. solium in the liver of pigs with naturally acquired porcine cysticercosis, as well as some T. solium infection in the lungs and spleen of some animals. We investigated the cause of lesions in sites other than the muscle or brain in a total of 157 pigs from T. solium endemic regions of Uganda and Nepal which were subjected to extensive investigations at necropsy. Lesions which had the potential to be caused by T. solium were characterised by macroscopic and microscopic examination, histology as well as DNA characterisation by PCR-RFLP and sequencing. Lesions were confirmed as being caused by Taenia hydatigena (both viable and non-viable), by T. asiatica and Echinococcus granulosus (in Nepal) and nematode infections. No T. solium-related lesions or cysticerci were identified in any tissue other than muscle and brain. It is recommended that future evaluations of porcine cysticercosis in aberrant tissue locations include DNA analyses that take appropriate care to avoid the possibility of contamination of tissue specimens with DNA from a different tissue location or a different animal. The use of appropriate control samples to confirm the absence of cross-sample contamination is also recommended. [ABSTRACT FROM AUTHOR]

Background: Salmonella enterica serovar Enteritidis is a cause of both poultry- and egg-associated enterocolitis globally and bloodstream-invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa (sSA). Distinct, multi-drug resistant genotypes associated with iNTS disease in sSA have recently been described, often requiring treatment with fluoroquinolone antibiotics. In industrialised countries, antimicrobial use in poultry production has led to frequent fluoroquinolone resistance amongst globally prevalent enterocolitis-associated lineages. Methodology/Principal findings: Twenty seven S. Enteritidis isolates from patients with iNTS disease and two poultry isolates, collected between 2007 and 2015 in the Ashanti region of Ghana, were whole-genome sequenced. These isolates, notable for a high rate of diminished ciprofloxacin susceptibility (DCS), were placed in the phyletic context of 1,067 sequences from the Public Health England (PHE) S. Enteritidis genome database to understand whether DCS was associated with African or globally-circulating clades of S. Enteritidis. Analysis showed four of the major S. Enteritidis clades were represented, two global and two African. All thirteen DCS isolates, containing a single gyrA mutation at codon 87, belonged to a global PT4-like clade responsible for epidemics of poultry-associated enterocolitis. Apart from two DCS isolates, which clustered with PHE isolates associated with travel to Spain and Brazil, the remaining DCS isolates, including one poultry isolate, belonged to two monophyletic clusters in which gyrA 87 mutations appear to have developed within the region. Conclusions/Significance: Extensive phylogenetic diversity is evident amongst iNTS disease-associated S. Enteritidis in Ghana. Antimicrobial resistance profiles differed by clade, highlighting the challenges of devising empirical sepsis guidelines. The detection of fluoroquinolone resistance in phyletically-related poultry and human isolates is of major concern and surveillance and control measures within the region’s burgeoning poultry industry are required to protect a human population at high risk of iNTS disease. [ABSTRACT FROM AUTHOR]

The importance of Zika virus (ZIKV) has increased noticeably since the outbreak in the Americas in 2015, when the illness was associated with congenital disorders. Although there is evidence of sexual transmission of the virus, the mosquito Aedes aegypti is believed to be the main vector for transmission to humans. This species of mosquito has not only been found naturally infected with ZIKV, but also has been the subject of study in many vector competence assays that employ different strains of ZIKV around the world. In Argentina, the first case was reported in February 2016 and a total of 278 autochthonous cases have since been confirmed, however, ZIKV virus has not been isolated from any mosquito species yet in Argentina. In order to elucidate if Argentinian Ae. aegypti populations could be a possible vector of ZIKV, we conducted vector competence studies that involved a local strain of ZIKV from Chaco province, and a Venezuelan strain obtained from an imported case. For this purpose, Ae. aegypti adults from the temperate area of Argentina (Buenos Aires province) were fed with infected blood. Body, legs and saliva were harvested and tested by plaque titration on plates of Vero cells for ZIKV at 7, 11 and 14 days post infection (DPI) in order to calculate infection, transmission, and dissemination rates, respectively. Both strains were able to infect mosquitoes at all DPIs, whereas dissemination and transmission were observed at all DPIs for the Argentinian strain but only at 14 DPI for the Venezuelan strain. This study proves the ability of Ae. aegypti mosquitoes from Argentina to become infected with two different strains of ZIKV, both belonging to the Asian lineage, and that the virus can disseminate to the legs and salivary glands. [ABSTRACT FROM AUTHOR]

Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas. [ABSTRACT FROM AUTHOR]

Oral cholera vaccines (OCVs) are being increasingly employed, but current killed formulations generally require multiple doses and lack efficacy in young children. We recently developed a new live-attenuated OCV candidate (HaitiV) derived from a Vibrio cholerae strain isolated during the 2010 Haiti cholera epidemic. HaitiV exhibited an unexpected probiotic-like activity in infant rabbits, preventing intestinal colonization and disease by wild-type V. cholerae before the onset of adaptive immunity. However, it remained unknown whether HaitiV would behave similarly to other OCVs to stimulate adaptive immunity against V. cholerae. Here, we orally immunized adult germ-free female mice to test HaitiV’s immunogenicity. HaitiV safely and stably colonized vaccinated mice and induced known adaptive immune correlates of cholera protection within 14 days of administration. Pups born to immunized mice were protected against lethal challenges of both homologous and heterologous V. cholerae strains. Cross-fostering experiments revealed that protection was not dependent on vaccine colonization in or transmission to the pups. These findings demonstrate the protective immunogenicity of HaitiV and support its development as a new tool for limiting cholera. [ABSTRACT FROM AUTHOR]

Despite worldwide mass drug administration, it is estimated that 68 million individuals are still infected with lymphatic filariasis with 19 million hydrocele and 17 million lymphedema reported cases. Despite the staggering number of pathology cases, the majority of LF-infected individuals do not develop clinical symptoms and present a tightly regulated immune system characterized by higher frequencies of regulatory T cells (Treg), suppressed proliferation and Th2 cytokine responses accompanied with increased secretion of IL-10, TGF-β and infection-specific IgG4. Nevertheless, the filarial-induced modulation of the host`s immune system and especially the role of regulatory immune cells like regulatory B (Breg) and Treg during an ongoing LF infection remains unknown. Thus, we analysed Breg and Treg frequencies in peripheral blood from Ghanaian uninfected endemic normals (EN), lymphedema (LE), asymptomatic patent (CFA+MF+) and latent (CFA+MF-) W. bancrofti-infected individuals as well as individuals who were previously infected with W. bancrofti (PI) but had cleared the infection due to the administration of ivermectin (IVM) and albendazole (ALB). In summary, we observed that IL-10-producing CD19+CD24highCD38dhigh Breg were specifically increased in patently infected (CFA+MF+) individuals. In addition, CD19+CD24highCD5+CD1dhigh and CD19+CD5+CD1dhighIL-10+ Breg as well as CD4+CD127-FOXP3+ Treg frequencies were significantly increased in both W. bancrofti-infected cohorts (CFA+MF+ and CFA+MF-). Interestingly, the PI cohort presented frequency levels of all studied regulatory immune cell populations comparable with the EN group. In conclusion, the results from this study show that an ongoing W. bancrofti infection induces distinct Breg and Treg populations in peripheral blood from Ghanaian volunteers. Those regulatory immune cell populations might contribute to the regulated state of the host immune system and are probably important for the survival and fertility (microfilaria release) of the helminth. [ABSTRACT FROM AUTHOR]

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics. [ABSTRACT FROM AUTHOR]

Background: Wolbachia’s ability to restrict arbovirus transmission makes it a promising tool to combat mosquito-transmitted diseases. Wolbachia-infected Aedes aegypti are currently being released in locations such as Brazil, which regularly experience concurrent outbreaks of different arboviruses. A. aegypti can become co-infected with, and transmit multiple arboviruses with one bite, which can complicate patient diagnosis and treatment. Methodology/principle findings: Using experimental oral infection of A. aegypti and then RT-qPCR, we examined ZIKV/DENV-1 and ZIKV/DENV-3 co-infection in Wolbachia-infected A. aegypti and observed that Wolbachia-infected mosquitoes experienced lower prevalence of infection and viral load than wildtype mosquitoes, even with an extra infecting virus. Critically, ZIKV/DENV co-infection had no significant impact on Wolbachia’s ability to reduce viral transmission. Wolbachia infection also strongly altered expression levels of key immune genes Defensin C and Transferrin 1, in a virus-dependent manner. Conclusions/significance: Our results suggest that pathogen interference in Wolbachia-infected A. aegypti is not adversely affected by ZIKV/DENV co-infection, which suggests that Wolbachia-infected A. aegypti will likely prove suitable for controlling mosquito-borne diseases in environments with complex patterns of arbovirus transmission. [ABSTRACT FROM AUTHOR]

Background: A single co-administered dose of ivermectin (IVM) plus diethylcarbamazine (DEC) plus albendazole (ALB), or triple-drug therapy, was recently found to be more effective for clearing microfilariae (Mf) than standard DEC plus ALB currently used for mass drug administration programs for lymphatic filariasis (LF) outside of sub-Saharan Africa. Triple-drug therapy has not been previously tested in LF-uninfected individuals from Africa. This study evaluated the pharmacokinetics (PK), safety, and efficacy of triple-drug therapy in people with and without Wuchereria bancrofti infection in West Africa. Methods: In this open-label cohort study, treatment-naïve microfilaremic (>50 mf/mL, n = 32) and uninfected (circulating filarial antigen negative, n = 24) adults residing in Agboville district, Côte d’Ivoire, were treated with a single dose of IVM plus DEC plus ALB, and evaluated for adverse events (AEs) until 7 days post treatment. Drug levels were assessed by liquid chromatography and mass spectrometry. Persons responsible for assessing AEs were blinded to participants’ infection status. Findings: There was no difference in AUC0-inf or Cmax between LF-infected and uninfected participants (P>0.05 for all comparisons). All subjects experienced mild AEs; 28% and 25% of infected and uninfected participants experienced grade 2 AEs, respectively. There were no severe or serious adverse events. Only fever (16 of 32 versus 4 of 24, P<0.001) and scrotal pain/swelling in males (6 of 20 versus 0 of 12, P = 0.025) were more frequent in infected than uninfected participants. All LF positive participants were amicrofilaremic at 7 days post-treatment and 27 of 31 (87%) remained amicrofilaremic 12 months after treatment. Conclusions: Moderate to heavy W. bancrofti infection did not affect PK parameters for IVM, DEC or ALB following a single co-administered dose of these drugs compared to uninfected individuals. The drugs were well tolerated. This study confirmed the efficacy of the triple-drug therapy for clearing W. bancrofti Mf and has added important information to support the use of this regimen in LF elimination programs in areas of Africa without co-endemic onchocerciasis or loiasis. Trial registration: ClinicalTrials.gov . [ABSTRACT FROM AUTHOR]

Background: The Mayaro virus (MAYV) is an endemic arbovirus in South American countries, where it is responsible for sporadic outbreaks of Mayaro fever. Clinical manifestations include fever, headache, ocular pain, rash, myalgia, and debilitating and persistent polyarthralgia. Understanding the mechanisms associated with MAYV-induced arthritis is of great importance due to the potential for its emergence, urbanization and dispersion to other regions. Methods: 15-day old Balb/c mice were infected by two distinct pathways, below the forelimb and in the rear footpad. Animals were observed for a period of 21 days. During this time, they were monitored every 24 hours for disease signs, such as weight loss and muscle weakness. Histological damage in the muscles and joints was evaluated 3, 7, 10, 15 and 20 days post-infection. The cytokine profile in serum and muscles during MAYV infection was evaluated by flow cytometry at different post-infection times. For pain analysis, the animals were submitted to the von Frey test and titre in different organs was evaluated throughout the study to obtain viral kinetics. Findings: Infection by two distinct pathways, below the forelimb and in the rear footpad, resulted in a homogeneous viral spread and the development of acute disease in animals. Clinical signs were observed such as ruffled fur, hunched posture, eye irritation and slight gait alteration. In the physical test, both groups presented loss of resistance, which was associated with histopathological damage, including myositis, arthritis, tenosynovitis and periostitis. The immune response was characterized by a strong inflammatory response mediated by the cytokines TNF-α, IL-6 and INF-γ and chemokine MCP-1, followed by the action of IL-10 and IL-4 cytokines. Interpretation: The results showed that Balb/c mice represent a promising model to study mechanisms involved in MAYV pathogenesis and for future antiviral testing. [ABSTRACT FROM AUTHOR]

Mosquitoes act as vectors of numerous pathogens that cause human diseases. Dengue virus (DENV) transmitted by mosquito, Aedes aegypti, is responsible for dengue fever epidemics worldwide with a serious impact on human health. Currently, disease control mainly relies on vector targeted intervention strategies. Therefore, it is imperative to understand the molecular mechanisms underlying the innate immune response of mosquitoes against pathogens. In the present study, the expression profiles of immunity-related genes in the midgut responding to DENV infection by feeding were analyzed by transcriptome and quantitative real-time PCR. The level of Antimicrobial peptides (AMPs) increased seven days post-infection (d.p.i.), which could be induced by the Toll immune pathway. The expression of reactive oxygen species (ROS) genes, including antioxidant genes, such as HPX7, HPX8A, HPX8B, HPX8C were induced at one d.p.i. and peaked again at ten d.p.i. in the midgut. Interestingly, down-regulation of the antioxidant gene HPX8C by RNA interference led to reduction in the virus titer in the mosquito, probably due to the elevated levels of ROS. Application of a ROS inhibitor and scavenger molecules further established the role of oxygen free radicals in the modulation of the immune response to DENV infection. Overall, our comparative transcriptome analyses provide valuable information about the regulation of immunity related genes in the transmission vector in response to DENV infection. It further allows us to identify novel molecular mechanisms underlying the host-virus interaction, which might aid in the development of novel strategies to control mosquito-borne diseases. [ABSTRACT FROM AUTHOR]

Leptospirosis is a zoonosis with a worldwide distribution, caused by pathogenic spirochetes of the genus Leptospira. The classification and identification of leptospires can be conducted by both genotyping and serotyping which are time-consuming and established in few reference laboratories. This study used matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as rapid and accurate tool for the identification of leptospires. The whole cell protein spectra of 116 Leptospira isolates including 15 references Leptospira spp. (pathogenic, n = 8; intermediate, n = 2; non-pathogenic, n = 5) and 101 Leptospira spp. clinical isolates was created as an in-house MALDI-TOF MS database. Ninety-seven clinical isolates from Thailand and Laos was validated with these protein spectra and revealed 98.9% correct identification when compared with 16S rRNA gene sequences method. Moreover, MALDI-TOF MS could identify spiked leptospires whole cell in urine. Biomarkers for differentiation of leptospires phylogeny and specific protein spectra for most found Leptospira spp. in this area (L. interrogans, L. kirschneri, L. borgpetersenii) based on MALDI-MS algorithm were demonstrated. [ABSTRACT FROM AUTHOR]

Background: Recent epidemics of Zika virus (ZIKV) in the Pacific and the Americas have highlighted its potential as an emerging pathogen of global importance. Both Aedes (Ae.) aegypti and Ae. albopictus are known to transmit ZIKV but variable vector competence has been observed between mosquito populations from different geographical regions and different virus strains. Since Australia remains at risk of ZIKV introduction, we evaluated the vector competence of local Ae. aegypti and Ae. albopictus for a Brazilian epidemic ZIKV strain. In addition, we evaluated the impact of daily temperature fluctuations around a mean of 28°C on ZIKV transmission and extrinsic incubation period. Methodology/Principal findings: Mosquitoes were orally challenged with a Brazilian ZIKV strain (8.8 log CCID50/ml) and maintained at either 28°C constant or fluctuating temperature conditions. At 3, 7 and 14 days post-infection (dpi), ZIKV RNA copies were quantified in mosquito bodies, as well as wings and legs, using qRT-PCR, while virus antigen in saliva (a proxy for transmission) was detected using a cell culture ELISA. Despite high body and disseminated infection rates in both vectors, the transmission rates of ZIKV in saliva of Ae. aegypti (50–60%) were significantly higher than in Ae. albopictus (10%) at 14 dpi. Both species supported a high viral load in bodies, with no significant differences between constant and fluctuating temperature conditions. However, a significant difference in viral load in wings and legs between species was observed, with higher titres in Ae. aegypti maintained at constant temperature conditions. For ZIKV transmission to occur in Ae. aegypti, a disseminated virus load threshold of 7.59 log10 copies had to be reached. Conclusions/Significance: Australian Ae. aegypti are better able to transmit a Brazilian ZIKV strain than Ae. albopictus. The results are in agreement with the global consensus that Ae. aegypti is the major vector of ZIKV. [ABSTRACT FROM AUTHOR]

Background: Clonorchis sinensis is a group I bio-carcinogen responsible for cholangiocarcinoma (CHCA) in humans. However, the mechanism by which C. sinensis promotes carcinogenesis is unclear. Methodology: Using the human cholangiocyte line H69, we investigated cell proliferation and gap junction protein expression after stimulation with the hepatotoxin N-nitrosodimethylamine (NDMA) and/or excretory-secretory products (ESP) of C. sinensis, which induce inflammation. NDMA and ESP treatment increased proliferation by 146% and the proportion of cells in the G2/M phase by 37%. Moreover, the expression of the cell proliferation-related proteins E2F1, Ki-67, and cancer related protein cytokeratin 19 and Cox-2 increased in response to combined treatment with NDMA and ESP. The gap-junction proteins connexin (Cx) 43 and Cx26 increased. In contrast, Cx32 expression decreased in cells treated with NDMA and ESP. Silencing of Cx43 reduced cell proliferation and significantly suppressed Cx26 and Cox-2 expression. Conclusions: These results suggest that Cx43 is an important factor in CHCA induced by C. sinensis ESP and NDMA and further investigations targeting this pathway may allow prevention of this deadly disease. [ABSTRACT FROM AUTHOR]

Young children are infected by a diverse variety of enteric pathogens in low-income, high-burden countries. Little is known about which conditions pose the greatest risk for enteric pathogen exposure and infection. Young children frequently play in residential public areas around their household, including areas contaminated by human and animal feces, suggesting these exposures are particularly hazardous. The objective of this study was to examine how the dose of six types of common enteric pathogens, and the probability of exposure to one or multiple enteric pathogens for young children playing at public play areas in Kisumu, Kenya is influenced by the type and frequency of child play behaviors that result in ingestion of soil or surface water. Additionally, we examine how pathogen doses and multi-pathogen exposure are modified by spatial variability in the number of public areas children are exposed to in their neighborhood. A Bayesian framework was employed to obtain the posterior distribution of pathogen doses for a certain number of contacts. First, a multivariate mixed effects tobit model was used to obtain the posterior distribution of pathogen concentrations, and their interdependencies, in soil and surface water, based upon empirical data of enteric pathogen contamination in three neighborhoods of Kisumu. Then, exposure doses were estimated using behavioral contact parameters from previous studies and contrasted under different exposure conditions. Pathogen presence and concentration in soil varied widely across local (< 25 meter radius area) and neighborhood-level scales, but pathogens were correlated among distinct surface water samples collected near to each other. Multi-pathogen exposure of children at public play areas was common. Pathogen doses and the probability of multi-pathogen ingestion increased with: higher frequency of environmental contact, especially for surface water; larger volume of soil or water ingested; and with play at multiple sites in the neighborhood versus single site play. Child contact with surface water and soil at public play areas in their neighborhood is an important cause of exposure to enteric pathogens in Kisumu, and behavioral, environmental, and spatial conditions are determinants of exposure. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus (RVFV) is a zoonotic phlebovirus that can be transmitted to humans or livestock by mosquitoes or through direct contact with contaminated bodily fluids and tissues. Exposure to bodily fluids and tissues varies by types of behaviors engaged for occupational tasks, homestead responsibilities, or use in dietary or therapeutic capacities. While previous studies have included milk exposures in their analyses, their primary focus on livestock exposures has been on animal handling, breeding, and slaughter. We analyzed data from multiple field surveys in Kenya with the aim of associating RVFV infection to raw milk exposures from common animal species. Of those with evidence of prior RVFV infection by serology (n = 267), 77.2% engaged in milking livestock compared to 32.0% for 3,956 co-local seronegative individuals (p < 0.001), and 86.5% of seropositive individuals consumed raw milk compared to 33.4% seronegative individuals (p < 0.001). Individuals who milked and also consumed raw milk had greater odds of RVFV exposure than individuals whose only contact to raw milk was through milking. Increased risks were associated with exposure to milk sourced from cows (p < 0.001), sheep (p < 0.001), and goats (p < 0.001), but not camels (p = 0.98 for consuming, p = 0.21 for milking). Our data suggest that exposure to raw milk may contribute to a significant number of cases of RVFV, especially during outbreaks and in endemic areas, and that some animal species may be associated with a higher risk for RVFV exposure. Livestock trade is regulated to limit RVFV spread from endemic areas, yet further interventions designed to fully understand the risk of RVFV exposure from raw milk are imperative. [ABSTRACT FROM AUTHOR]

Laboratory confirmation of Zika virus (ZIKV) infection during pregnancy is challenging due to cross-reactivity with dengue virus (DENV) and limited knowledge about the kinetics of anti-Zika antibody responses during pregnancy. We described ZIKV and DENV serological markers and the maternal-fetal transfer of antibodies among mothers and neonates after the ZIKV microcephaly outbreak in Northeast Brazil (2016). We included 89 microcephaly cases and 173 neonate controls at time of birth and their mothers. Microcephaly cases were defined as newborns with a particular head circumference (2 SD below the mean). Two controls without microcephaly were matched by the expected date of delivery and area of residence. We tested maternal serum for recent (ZIKV genome, IgM and IgG3 anti-NS1) and previous (ZIKV and DENV neutralizing antibodies [NAbs]) markers of infection. Multiple markers of recent or previous ZIKV and DENV infection in mothers were analyzed using principal component analysis (PCA). At delivery, 5.6% of microcephaly case mothers and 1.7% of control mothers were positive for ZIKV IgM. Positivity for ZIKV IgG3 anti-NS1 was 8.0% for case mothers and 3.5% for control mothers. ZIKV NAbs was slightly higher among mothers of cases (69.6%) than that of mothers of controls (57.2%; p = 0.054). DENV exposure was detected in 85.8% of all mothers. PCA discriminated two distinct components related to recent or previous ZIKV infection and DENV exposure. ZIKV NAbs were higher in newborns than in their corresponding mothers (p<0.001). We detected a high frequency of ZIKV exposure among mothers after the first wave of the ZIKV outbreak in Northeast Brazil. However, we found low sensitivity of the serological markers to recent infection (IgM and IgG3 anti-NS1) in perinatal samples of mothers of microcephaly cases. Since the neutralization test cannot precisely determine the time of infection, testing for ZIKV immune status should be performed as early as possible and throughout pregnancy to monitor acute Zika infection in endemic areas. [ABSTRACT FROM AUTHOR]

Coral snakes of the genus Micrurus have a high diversity and a wide distribution in the Americas. Despite envenomings by these animals are uncommon, accidents are often severe and may result in death. Producing an antivenom to treat these envenomings has been challenging since coral snakes are difficult to catch, produce small amounts of venom, and the antivenoms produced have shown limited cross neutralization. Here we present data of cross neutralization among monovalent antivenoms raised against M. dumerilii, M. isozonus, M. mipartitus and M. surinamensis and the development of a new polyvalent coral snake antivenom, resulting from the mix of monovalent antivenoms. Our results, show that this coral snake antivenom has high neutralizing potency and wide taxonomic coverage, constituting a possible alternative for a long sought Pan-American coral snake antivenom. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a mosquito-borne positive sense RNA virus. Recently, ZIKV emerged into the Western hemisphere as a human health threat, with severe disease associated with developmental and neurological complications. The structural envelope protein of ZIKV and other neurotropic flaviviruses contains an extended CD-loop relative to non-neurotropic flaviviruses, and has been shown to augment ZIKV stability and pathogenesis. Here we show that shortening the CD-loop in ZIKV attenuates the virus in mice, by reducing the ability to invade and replicate in the central nervous system. The CD-loop mutation was genetically stable following infection in mice, though secondary site mutations arise adjacent to the CD-loop. Importantly, while shortening of the CD-loop attenuates the virus, the CD-loop mutant maintains antigenicity in immunocompetent mice, eliciting an antibody response that similarly neutralizes both the mutant and wildtype ZIKV. These findings suggest that the extended CD-loop in ZIKV is a determinant of neurotropism and may be a target in live-attenuated vaccine design, for not only ZIKV, but for other neurotropic flaviviruses. [ABSTRACT FROM AUTHOR]