A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host.

Chemical synthesis of conjugate vaccines, consisting of a polysaccharide linked to a protein, can be technically challenging, and in vivo bacterial conjugations (bioconjugations) have emerged as manufacturing alternatives. Bioconjugation relies upon an oligosaccharyltransferase to attach polysaccharides to proteins, but currently employed enzymes are not suitable for the generation of conjugate vaccines when the polysaccharides contain glucose at the reducing end, which is the case for ~75% of Streptococcus pneumoniae capsules. Here, we use an O-linking oligosaccharyltransferase to generate a polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end. In addition, we show that different vaccine carrier proteins can be glycosylated using this system. Pneumococcal bioconjugates are immunogenic, protective and rapidly produced within E. coli using recombinant techniques. These proof-of-principle experiments establish a platform to overcome limitations of other conjugating enzymes enabling the development of bioconjugate vaccines for many important human and animal pathogens.

Bat Caliciviruses and Human Noroviruses Are Antigenically Similar and Have Overlapping Histo-Blood Group Antigen Binding Profiles.

Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens.IMPORTANCE Caliciviruses are rapidly evolving viruses that cause pandemic outbreaks associated with significant morbidity and mortality globally. The animal reservoirs for human caliciviruses are unknown; bats represent critical reservoir species for several emerging and zoonotic diseases. Recent reports have identified several bat caliciviruses but have not characterized biological functions associated with disease risk, including their potential emergence in other mammalian populations. In this report, we identified a novel bat calicivirus that is most closely related to nonhuman primate caliciviruses. Using this new bat calicivirus and a second norovirus-like bat calicivirus capsid gene sequence, we generated virus-like particles that have host carbohydrate ligand binding patterns similar to those of human and animal noroviruses and that share antigens with human noroviruses. The similarities to human noroviruses with respect to binding patterns and antigenic epitopes illustrate the potential for bat caliciviruses to emerge in other species and the importance of pathogen surveillance in wild-animal populations.

Bhlhe40 is an essential repressor of IL-10 during Mycobacterium tuberculosis infection.

The cytokine IL-10 antagonizes pathways that control Mycobacterium tuberculosis (Mtb) infection. Nevertheless, the impact of IL-10 during Mtb infection has been difficult to decipher because loss-of-function studies in animal models have yielded only mild phenotypes. We have discovered that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) is required to repress Il10 expression during Mtb infection. Loss of Bhlhe40 in mice results in higher Il10 expression, higher bacterial burden, and early susceptibility similar to that observed in mice lacking IFN-γ. Deletion of Il10 in Bhlhe40-/- mice reverses these phenotypes. Bhlhe40 deletion in T cells or CD11c+ cells is sufficient to cause susceptibility to Mtb Bhlhe40 represents the first transcription factor found to be essential during Mtb infection to specifically regulate Il10 expression, revealing the importance of strict control of IL-10 production by innate and adaptive immune cells during infection. Our findings uncover a previously elusive but significant role for IL-10 in Mtb pathogenesis.

LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

Irg1 expression in myeloid cells prevents immunopathology during M. tuberculosis infection.

Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions, principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through its inhibitory effects on cytokine and reactive oxygen species production. To evaluate the functions of Irg1 in vivo, we challenged wild-type (WT) and Irg1-/- mice with Mycobacterium tuberculosis (Mtb) and monitored disease progression. Irg1-/-, but not WT, mice succumbed rapidly to Mtb, and mortality was associated with increased infection, inflammation, and pathology. Infection of LysM-Cre Irg1fl/fl, Mrp8-Cre Irg1fl/fl, and CD11c-Cre Irg1fl/fl conditional knockout mice along with neutrophil depletion experiments revealed a role for Irg1 in LysM+ myeloid cells in preventing neutrophil-mediated immunopathology and disease. RNA sequencing analyses suggest that Irg1 and its production of itaconate temper Mtb-induced inflammatory responses in myeloid cells at the transcriptional level. Thus, an Irg1 regulatory axis modulates inflammation to curtail Mtb-induced lung disease.

Exploring dental student participation in interdisciplinary care team conferences in long-term care.

OBJECTIVE: The purpose of this study was to explore how the participation of dental hygiene students in interdisciplinary care conferences conducted in long-term care (LTC) facilities influenced the staff's awareness of oral health, the student's provision of care as well as the student's ability to work on an interdisciplinary team. BACKGROUND: Oral health in LTC continues to be poor despite many educational and clinical interventions. The care of a frail elder requires an interdisciplinary team approach, yet dental professionals are often absent from these teams. As an educational intervention, dental hygiene students, in addition to providing clinical care to residents and education to staff in a LTC facility, have been participating in interdisciplinary care conferences for 4 years. METHODS: Nine LTC facility staff and eight students participated in two focus groups and five personal interviews to discuss their experiences of the care conferences. Narratives were transcribed verbatim and thematic analysis was conducted. RESULTS: Four major themes emerged that described the essence of the experience of involving dental professionals within interdisciplinary care conference: (i) addressing oral health, (ii) reciprocal learning, (iii) dealing with complexity and (iv) impact of collaborative care. CONCLUSION: Overall, students found the educational intervention to be worthwhile that allowed for the development of interdisciplinary skills and a greater understanding of dental geriatrics. The dental support in care conferences was welcomed by the staff as it facilitated a new way of exchanging knowledge and experiences so that a more holistic approach to care could be undertaken by all.

Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence.

CarD is an essential RNA polymerase (RNAP) interacting protein in Mycobacterium tuberculosis that stimulates formation of RNAP-promoter open complexes. CarD plays a complex role in M. tuberculosis growth and virulence that is not fully understood. Therefore, to gain further insight into the role of CarD in M. tuberculosis growth and virulence, we determined the effect of increasing the affinity of CarD for RNAP. Using site-directed mutagenesis guided by crystal structures of CarD bound to RNAP, we identified amino acid substitutions that increase the affinity of CarD for RNAP. Using these substitutions, we show that increasing the affinity of CarD for RNAP increases the stability of the CarD protein in M. tuberculosis In addition, we show that increasing the affinity of CarD for RNAP increases the growth rate in M. tuberculosis without affecting 16S rRNA levels. We further show that increasing the affinity of CarD for RNAP reduces M. tuberculosis virulence in a mouse model of infection despite the improved growth rate in vitro Our findings suggest that the CarD-RNAP interaction protects CarD from proteolytic degradation in M. tuberculosis, establish that growth rate and rRNA levels can be uncoupled in M. tuberculosis and demonstrate that the strength of the CarD-RNAP interaction has been finely tuned to optimize virulence. IMPORTANCE: Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health problem. In order to develop new strategies to battle this pathogen, we must gain a better understanding of the molecular processes involved in its survival and pathogenesis. We have previously identified CarD as an essential transcriptional regulator in mycobacteria. In this study, we detail the effects of increasing the affinity of CarD for RNAP on transcriptional regulation, CarD protein stability, and virulence. These studies expand our understanding of the global transcription regulator CarD, provide insight into how CarD activity is regulated, and broaden our understanding of prokaryotic transcription.

Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates.

We describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2-7 days post-challenge. All naïve macaques had detectable viral RNA from day 2-10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10-30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further development of a tetravalent EDIII-E2 dengue vaccine.

Unique role for ATG5 in neutrophil-mediated immunopathology during M. tuberculosis infection.

Mycobacterium tuberculosis, a major global health threat, replicates in macrophages in part by inhibiting phagosome-lysosome fusion, until interferon-γ (IFNγ) activates the macrophage to traffic M. tuberculosis to the lysosome. How IFNγ elicits this effect is unknown, but many studies suggest a role for macroautophagy (herein termed autophagy), a process by which cytoplasmic contents are targeted for lysosomal degradation. The involvement of autophagy has been defined based on studies in cultured cells where M. tuberculosis co-localizes with autophagy factors ATG5, ATG12, ATG16L1, p62, NDP52, BECN1 and LC3 (refs 2-6), stimulation of autophagy increases bacterial killing, and inhibition of autophagy increases bacterial survival. Notably, these studies reveal modest (~1.5-3-fold change) effects on M. tuberculosis replication. By contrast, mice lacking ATG5 in monocyte-derived cells and neutrophils (polymorponuclear cells, PMNs) succumb to M. tuberculosis within 30 days, an extremely severe phenotype similar to mice lacking IFNγ signalling. Importantly, ATG5 is the only autophagy factor that has been studied during M. tuberculosis infection in vivo and autophagy-independent functions of ATG5 have been described. For this reason, we used a genetic approach to elucidate the role for multiple autophagy-related genes and the requirement for autophagy in resistance to M. tuberculosis infection in vivo. Here we show that, contrary to expectation, autophagic capacity does not correlate with the outcome of M. tuberculosis infection. Instead, ATG5 plays a unique role in protection against M. tuberculosis by preventing PMN-mediated immunopathology. Furthermore, while Atg5 is dispensable in alveolar macrophages during M. tuberculosis infection, loss of Atg5 in PMNs can sensitize mice to M. tuberculosis. These findings shift our understanding of the role of ATG5 during M. tuberculosis infection, reveal new outcomes of ATG5 activity, and shed light on early events in innate immunity that are required to regulate disease pathology and bacterial replication.

A simian hemorrhagic fever virus isolate from persistently infected baboons efficiently induces hemorrhagic fever disease in Japanese macaques.

Simian hemorrhagic fever virus is an arterivirus that naturally infects species of African nonhuman primates causing acute or persistent asymptomatic infections. Although it was previously estimated that 1% of baboons are SHFV-positive, more than 10% of wild-caught and captive-bred baboons tested were SHFV positive and the infections persisted for more than 10 years with detectable virus in the blood (100-1000 genomes/ml). The sequences of two baboon SHFV isolates that were amplified by a single passage in primary macaque macrophages had a high degree of identity to each other as well as to the genome of SHFV-LVR, a laboratory strain isolated in the 1960s. Infection of Japanese macaques with 100PFU of a baboon isolate consistently produced high level viremia, pro-inflammatory cytokines, elevated tissue factor levels and clinical signs indicating coagulation defects. The baboon virus isolate provides a reliable BSL2 model of viral hemorrhagic fever disease in macaques.

RNA populations in immunocompromised patients as reservoirs for novel norovirus variants.

UNLABELLED: Noroviruses are the leading cause of acute gastroenteritis outbreaks worldwide. The majority of norovirus outbreaks are caused by genogroup II.4 (GII.4). Novel GII.4 strains emerge every 2 to 4 years and replace older variants as the dominant norovirus. Novel variants emerge through a combination of recombination, genetic drift, and selection driven by population immunity, but the exact mechanism of how or where is not known. We detected two previously unknown novel GII.4 variants, termed GII.4 UNK1 and GII.4 UNK2, and a diverse norovirus population in fecal specimens from immunocompromised individuals with diarrhea after they had undergone bone marrow transplantation. We hypothesized that immunocompromised individuals can serve as reservoirs for novel norovirus variants. To test our hypothesis, metagenomic analysis of viral RNA populations was combined with a full-genome bioinformatic analysis of publicly available GII.4 norovirus sequences from 1974 to 2014 to identify converging sites. Variable sites were proportionally more likely to be within two amino acids (P < 0.05) of positively selected sites. Further analysis using a hypergeometric distribution indicated that polymorphic site distribution was random and its proximity to positively selected sites was dependent on the size of the norovirus genome and the number of positively selected sites.In conclusion, random mutations may have a positive impact on driving norovirus evolution, and immunocompromised individuals could serve as potential reservoirs for novel GII.4 strains. IMPORTANCE: Norovirus is the most common cause of viral gastroenteritis in the United States. Every 2 to 3 years novel norovirus variants emerge and replace dominant strains. The continual emergence of novel noroviruses is believed to be caused by a combination of genetic drift, population immunity, and recombination, but exactly how this emergence occurs remains unknown. In this study, we identified two novel GII.4 variants in immunocompromised bone marrow transplant patients. Using metagenomic and bioinformatic analysis, we showed that most genetic polymorphisms in the novel variants occur near 0 to 2 amino acids of positively selected sites, but the distribution of mutations was random; clustering of polymorphisms with positively selected sites was a result of genome size and number of mutations and positively selected sites. This study shows that immunocompromised patients can harbor infectious novel norovirus variants, and although mutations in viruses are random, they can have a positive effect on viral evolution.

A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant.

Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (ß-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c ß-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c ß-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. IMPORTANCE The 2012 outbreak of MERS-CoV raises the specter of another global epidemic, similar to the 2003 SARS-CoV epidemic. MERS-CoV is related to BtCoV HKU5 in target regions that are essential for drug and vaccine testing. Because no small animal model exists to evaluate MERS-CoV pathogenesis or to test vaccines, we constructed a recombinant BtCoV HKU5 that expressed a region of the SARS-CoV spike (S) glycoprotein, thereby allowing the recombinant virus to grow in cell culture and in mice. We show that this recombinant virus targets airway epithelial cells and causes disease in aged mice. We use this platform to (i) identify a broad-spectrum antiviral that can potentially inhibit viruses closely related to MERS-CoV, (ii) demonstrate the absence of increased eosinophilic immune pathology for MERS-CoV N protein-based vaccines, and (iii) mouse adapt this virus to identify viral genetic determinants of cross-species transmission and virulence. This study holds significance as a strategy to control newly emerging viruses.

Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity.

The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens.

The mechanism of differential neutralization of dengue serotype 3 strains by monoclonal antibody 8A1.

While previous studies have demonstrated that envelope (E) glycoprotein variation between dengue viruses (DENV) genotypes can influence antibody neutralization potency, the mechanisms of variable neutralization remain incompletely understood. Here we characterize epitope antibody interactions of a DENV-3 EDIII binding mouse mAb 8A1 which displays highly variable neutralizing activity against DENV-3 genotypes. Using a DENV-3 reverse genetics platform, we characterize ability of 8A1 to bind and neutralize naturally occurring DENV-3 E genotypic variant viruses. Introduction of single and multiple amino acid mutations into the parental clone background demonstrates that mutations at positions 301 and 383 on EDIII are responsible for 8A1 differential neutralization phenotypes. ELISA and surface plasmon resonance (SPR) studies indicate differences in binding are responsible for the variable neutralization. Variability at position 301 primarily determined binding difference through influencing antibody-EDIII dissociation rate. Our findings are relevant to many groups focusing on DENV EDIII as a vaccine target.

Evidence supporting a zoonotic origin of human coronavirus strain NL63.

The relationship between bats and coronaviruses (CoVs) has received considerable attention since the severe acute respiratory syndrome (SARS)-like CoV was identified in the Chinese horseshoe bat (Rhinolophidae) in 2005. Since then, several bats throughout the world have been shown to shed CoV sequences, and presumably CoVs, in the feces; however, no bat CoVs have been isolated from nature. Moreover, there are very few bat cell lines or reagents available for investigating CoV replication in bat cells or for isolating bat CoVs adapted to specific bat species. Here, we show by molecular clock analysis that alphacoronavirus (α-CoV) sequences derived from the North American tricolored bat (Perimyotis subflavus) are predicted to share common ancestry with human CoV (HCoV)-NL63, with the most recent common ancestor between these viruses occurring approximately 563 to 822 years ago. Further, we developed immortalized bat cell lines from the lungs of this bat species to determine if these cells were capable of supporting infection with HCoVs. While SARS-CoV, mouse-adapted SARS-CoV (MA15), and chimeric SARS-CoVs bearing the spike genes of early human strains replicated inefficiently, HCoV-NL63 replicated for multiple passages in the immortalized lung cells from this bat species. These observations support the hypothesis that human CoVs are capable of establishing zoonotic-reverse zoonotic transmission cycles that may allow some CoVs to readily circulate and exchange genetic material between strains found in bats and other mammals, including humans.

Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions.

Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (mAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human mAbs derived from DENV-immune individuals. Two of three strongly neutralizing human mAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. We propose that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field.

Development and characterization of a reverse genetic system for studying dengue virus serotype 3 strain variation and neutralization.

Dengue viruses (DENV) are enveloped single-stranded positive-sense RNA viruses transmitted by Aedes spp. mosquitoes. There are four genetically distinct serotypes designated DENV-1 through DENV-4, each further subdivided into distinct genotypes. The dengue scientific community has long contended that infection with one serotype confers lifelong protection against subsequent infection with the same serotype, irrespective of virus genotype. However this hypothesis is under increased scrutiny and the role of DENV genotypic variation in protection from repeated infection is less certain. As dengue vaccine trials move increasingly into field-testing, there is an urgent need to develop tools to better define the role of genotypic variation in DENV infection and immunity. To better understand genotypic variation in DENV-3 neutralization and protection, we designed and constructed a panel of isogenic, recombinant DENV-3 infectious clones, each expressing an envelope glycoprotein from a different DENV-3 genotype; Philippines 1982 (genotype I), Thailand 1995 (genotype II), Sri Lanka 1989 and Cuba 2002 (genotype III) and Puerto Rico 1977 (genotype IV). We used the panel to explore how natural envelope variation influences DENV-polyclonal serum interactions. When the recombinant viruses were tested in neutralization assays using immune sera from primary DENV infections, neutralization titers varied by as much as ∼19-fold, depending on the expressed envelope glycoprotein. The observed variability in neutralization titers suggests that relatively few residue changes in the E glycoprotein may have significant effects on DENV specific humoral immunity and influence antibody mediated protection or disease enhancement in the setting of both natural infection and vaccination. These genotypic differences are also likely to be important in temporal and spatial microevolution of DENV-3 in the background of heterotypic neutralization. The recombinant and synthetic tools described here are valuable for testing hypotheses on genetic determinants of DENV-3 immunopathogenesis.

Metagenomic analysis of the viromes of three North American bat species: viral diversity among different bat species that share a common habitat.

Effective prediction of future viral zoonoses requires an in-depth understanding of the heterologous viral population in key animal species that will likely serve as reservoir hosts or intermediates during the next viral epidemic. The importance of bats as natural hosts for several important viral zoonoses, including Ebola, Marburg, Nipah, Hendra, and rabies viruses and severe acute respiratory syndrome-coronavirus (SARS-CoV), has been established; however, the large viral population diversity (virome) of bats has been partially determined for only a few of the ∼1,200 bat species. To assess the virome of North American bats, we collected fecal, oral, urine, and tissue samples from individual bats captured at an abandoned railroad tunnel in Maryland that is cohabitated by 7 to 10 different bat species. Here, we present preliminary characterization of the virome of three common North American bat species, including big brown bats (Eptesicus fuscus), tricolored bats (Perimyotis subflavus), and little brown myotis (Myotis lucifugus). In samples derived from these bats, we identified viral sequences that were similar to at least three novel group 1 CoVs, large numbers of insect and plant virus sequences, and nearly full-length genomic sequences of two novel bacteriophages. These observations suggest that bats encounter and disseminate a large assortment of viruses capable of infecting many different animals, insects, and plants in nature.