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Dengue human infection models present an opportunity to explore a vaccine, antiviral, or immuno-compound’s potential for clinical benefit in a controlled setting. Herein, we report the outcome of a phase 1, open-label assessment of a DENV-3 challenge model. In this study, 9 participants received a subcutaneous inoculation with 0.5ml of a 1.4×103PFU/ml suspension of the DENV-3 strain CH53489. All subjects developed RNAemia within 7 days of inoculation, with peak titers ranging from 3.13×104to 7.02×108GE/ml. Symptoms and clinical lab abnormalities consistent with mild dengue infection were observed in all subjects. DENV-3 specific seroconversion was observed by 14 days after inoculation, along with DENV-3 specific memory T cell responses. RNAseq and serum cytokine analysis revealed the presence of an antiviral transcriptional and cytokine response to infection that overlapped with the period of viremia. The magnitude and frequency of clinical and immunologic endpoints correlated with an individual’s peak viral titer.

Influenza within-host viral populations are the source of all global influenza diversity and play an important role in driving the evolution and escape of the influenza virus from human immune responses, antiviral treatment, and vaccines, and have been used in precision tracking of influenza transmission chains. Next Generation Sequencing (NGS) has greatly improved our ability to study these populations, however, major challenges remain, such as accurate identification of intra-host single nucleotide variants (iSNVs) that represent within-host viral diversity of influenza virus. In order to investigate the sources and the frequency of called iSNVs in influenza samples, we used a set of longitudinal influenza patient samples collected within a University of Maryland (UMD) cohort of college students in a living learning community. Our results indicate that technical replicates aid in removal of random RT-PCR, PCR, and platform sequencing errors, while the use of clonal plasmids for removal of systematic errors is more important in samples of low RNA abundance. We show that the choice of reference for read mapping affects the frequency of called iSNVs, with the sample self-reference resulting in the lowest amount of iSNV noise. The importance of variant caller choice is also highlighted in our study, as we observe differential sensitivity of variant callers to the mapping reference choice, as well as the poor overlap of their called iSNVs. Based on this, we develop an approach for identification of highly probable iSNVs by removal of sequencing and bioinformatics algorithm-associated errors, which we implement in phylogenetic analyses of the UMD samples for a greater resolution of transmission links. In addition to identifying closely related transmission connections supported by the presence of highly confident shared iSNVs between patients, our results also indicate that the rate of minor variant turnover within a host may be a limiting factor for utilization of iSNVs to determine patient epidemiological links.

Recent outbreaks of emerging and re-emerging viruses such as Zika, West Nile and Japanese encephalitis (JEV) viruses have shown that timely detection of novel arboviruses with epidemic potential is essential to mitigate human health risks. There have been rising concerns that an emergent JEV genotype (genotype V, GV) is circulating in Asia, against which the current US-FDA-approved JEV vaccine may not be efficacious. To ascertain if JEV GV and other arboviruses are circulating in East Asia, we conducted next-generation sequencing on 260 pools of Culex tritaeniorhynchus and Culex bitaeniorhynchus mosquitoes (6,540 specimens) collected at Camp Humphreys, Republic of Korea (ROK), from mid-May - October 2018. Metagenomic analysis demonstrated a highly abundant and diverse virome with correlates of health and ecological relevance. Additionally, two complete JEV GV genome sequences were obtained from separate mosquito pools, indicating that JEV GV is circulating in the Pyeongtaek area near Seoul, ROK. Retrospective sample and sequence analyses showed that JEV GV was also present in 2016 mosquito pools collected in Seoul, ROK. Sequence-based analysis of JEV GV indicates a divergent genotype that is the most distant from the GIII derived live attenuated SA14-14-2 vaccine strain. A GV E protein investigation and 3D modeling in context to SA14-14-2 indicated likely regions responsible for reduced antibody affinity, including clusters of significant amino acid changes at externally exposed domains. These data highlight the critical need for continued mosquito surveillance as a means of detecting and identifying emerging and re-emerging arboviruses of public health relevance. Importantly, our results emphasize recent concerns that there may be a possible shift in the circulating JEV genotype in East Asia and highlights the critical need for a vaccine proven to be efficacious against this re-emergent virus.

For most pathogens, transmission is driven by interactions between the behaviours of infectious individuals, the behaviours of the wider population, the local environment, and immunity. Phylogeographic approaches are currently unable to disentangle the relative effects of these competing factors. We develop a spatiotemporally structured phylogenetic framework that reconstructs transmission across spatial scales and apply it to geocoded dengue virus sequences from Thailand (N=726 over 18 years). We find infected individuals spend 96% of their daytime hours in their home community compared to 76% for the susceptible population (mainly children) and 42% for adults. Dynamic pockets of local immunity make transmission more likely in places with high heterotypic immunity and less likely where high homotypic immunity exists. Age-dependent mixing of individuals and vector distributions are not important in determining transmission. This approach provides previously unknown insights into one of the most complex disease systems known and will be applicable across pathogens.

Natural dengue virus (DENV) infections are delivered by mosquito bite but how the route of inoculation route could shape the humoral immune response is not well understood. Here, we serologically profiled 20 non-human primates (NHP) from a prior study of DENV1 infection in which the animals were inoculated by mosquito (N=10) or subcutaneous injection (N=10). Using a comprehensive, densely tiled and highly redundant pan-flavivirus programmable phage library containing 91,562 overlapping 62 amino acid peptides, we produced a high-resolution map of linear peptide sequences enriched during DENV seroconversion. We found that serological profiles in mosquito-inoculated and subcutaneously-inoculated animals were similar up to 90 days after primary infection, but diverged at 1 year. We found differences in sero-reactivity, as indicated by the median area under the curve (AUC) in the Envelope (E; residues 215-406; p < 0.08), and Nonstructural-3 (NS3; residues 549-615; p < 0.05) proteins in mosquito-inoculated versus subcutaneously-inoculated animals. Within the E protein, residues 339-384 in domain III accounted for >99% of the total AUC difference across residues 215-406. Antibody breadth did not vary by mode of inoculation. The differential reactivity to E domain III (EDIII) seen by phage display validated orthogonally by ELISA, but did not correlate with late neutralization titers. Serological profiling of humoral immune responses to DENV infection in NHP by programmable phage display demonstrated durable differences in sero-reactivity by route of inoculation. These findings could have implications for DENV diagnostics and evaluation of vaccines.IMPORTANCEDengue virus (DENV) infections are transmitted by mosquito bite, but how being infected by mosquito bite affects the immune response is not known. In this study, we analyzed antibodies produced by rhesus macaques infected with DENV using programmable phage display, a high-throughput method for characterizing what viral protein derived peptides serum antibodies bind to. We found that while there was no difference in antibody binding profiles at early timepoints post-infection, at one year post-infection, there were substantial differences in the antibody binding profiles of macaques who were infected by mosquito bite versus those that were infected by injection. In general, antibodies in the macaques inoculated by mosquito maintained higher levels of sero-reactivity, with a strong signal still present one year post-infection. The findings we report could have implications for DENV diagnostics and evaluation of DENV vaccines.

SummaryThe global emergence of Zika virus (ZIKV) in the last decade revealed the unprecedented ability for a mosquito-borne virus to cause congenital birth defects such as microcephaly. A puzzling aspect of ZIKV emergence is that all human outbreaks and birth defects to date have been exclusively associated with the Asian ZIKV lineage, despite a growing body of laboratory evidence pointing towards higher transmissibility and pathogenicity of the African ZIKV lineage. Whether this apparent paradox reflects the use of relatively old African ZIKV strains in most laboratory studies is unclear. Here, we experimentally compared the transmissibility and pathogenicity of seven low-passage ZIKV strains representing the recently circulating viral genetic diversity. We found that recent African ZIKV strains largely outperformed their Asian counterparts in mosquito transmission kinetics experiments, which translated into a markedly higher epidemic potential in outbreak computer simulations. In addition, African ZIKV strains were significantly more lethal than Asian ZIKV strains in immunocompromised adult mice. Finally, prenatal infection of immunocompetent mouse embryos with an African ZIKV strain resulted in embryonic death whereas it caused microcephaly with Asian ZIKV strains. Together, our results demonstrate the high epidemic potential and pathogenicity of recent ZIKV strains from Africa. Importantly, they also imply that the African ZIKV lineage could more easily go unnoticed by public health surveillance systems than the Asian ZIKV lineage due to its propensity to cause fetal loss rather than birth defects.

Intra-host single nucleotide variants (iSNVs) have been increasingly used in genomic epidemiology to increase phylogenetic resolution and reconstruct fine-scale outbreak dynamics. These analyses are preferably done on sequence data from direct clinical samples, but in many cases due to low viral loads, there might not be enough genetic material for deep sequencing and iSNV determination. Isolation of the virus from clinical samples with low passage number increases viral load, but to date, no studies have investigated how dengue virus (DENV) culture isolation from a clinical sample impacts the consensus sequence, and there is no information on the intra-host virus population changes that may result from viral isolation. In this study, we investigate consensus and iSNV frequency differences between DENV sequenced directly from clinical samples and their corresponding low-passage isolates. Twenty five DENV1 and DENV2 positive sera and their corresponding viral isolates (T.splendens inoculation and C6/36 passage) were obtained from a prospective cohort study in the Philippines. These were sequenced on MiSeq with minimum nucleotide depth of coverage of 1000x, and iSNVs were detected using LoFreq. For both DENV1 and DENV2, we found that the nucleotide call concordance (including called iSNVs with variant cutoff at 5%) between direct sera sample and its cultured virus was on average 99.99%. There were a maximum of one consensus nucleotide difference between clinical sample and isolate. Interestingly, we found that iSNV frequencies were also largely preserved between the samples, with an average difference in minor variant frequency of 6.8% (95CI: 3.6%-10%) and 9.6% (95CI: 7%-12.2%) for DENV1 and DENV2, respectively. Furthermore, we found no significant differences in either DENV1 or DENV2 between the sample pairs (clinical sample and isolate) in their number of iSNV positions per genome, or in the difference in variant frequencies (p=0.36 and p=0.13, respectively, F-test). Our results show that low-passage DENV isolates may be used for identification of the majority of their human-derived within-host variant populations, which are increasingly being used for precision tracking of DENV and other RNA viruses.

Controlled dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To bridge this knowledge gap, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 controlled human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both controlled and natural DENV infection resulted in overlapping patterns of inflammatory gene upregulation, natural DENV infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by controlled and natural primary DENV infection are similar, but that natural DENV infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infectedin vitrowith DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5’ capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5’ to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5’ end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5’ capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naïve B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5’ capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells.IMPORTANCEDengue is one of the most widespread vector-borne viral diseases in the world. However, it is still unclear which cells harbor virus during DENV infection, and how viral reservoirs in serum and infected cells are related. These results demonstrate for the first time that intracellular DENV RNA can be identified and infected cells quantified by 5’ capture scRNAseq. This strategy provides a significantly greater throughput and higher sensitivity than previously published methods and has the potential to provide additional information on the genomic heterogeneity of intracellular DENV RNA.

Kenya experiences a substantial burden of dengue, yet there are very few DENV-2 sequence data available from this country and indeed the entire continent of Africa. We therefore undertook whole genome sequencing and evolutionary analysis of fourteen dengue virus (DENV)-2 strains sampled from Malindi sub-County Hospital during the 2017 DENV-2 outbreak in the Kenyan coast. We further performed an extended East African phylogenetic analysis, which leveraged 26 complete Africanenvgenes. Maximum likelihood analysis showed that the 2017 outbreak was due to the Cosmopolitan genotype, indicating that this has been the only confirmed human DENV-2 genotype circulating in Africa to date. Phylogeographic analyses indicated transmission of DENV-2 viruses between East Africa and South/South-West Asia. Time-scaled genealogies show that DENV-2 viruses are spatially structured within Kenya, with a time-to-most-common-recent ancestor analysis indicating that these DENV-2 strains were circulating for up to 5.38 years in Kenya before detection in the 2017 Malindi outbreak. Selection pressure analyses indicated sampled Kenyan DENV strains uniquely being under positive selection at 6 sites, predominantly across the non-structural genes, and epitope prediction analyses showed that one of these sites corresponds to a putative predicted MHC-I CD8+ DENV-2 Cosmopolitan virus epitope only evident in a sampled Kenyan virus. Taken together, our findings indicate that the 2017 Malindi DENV-2 outbreak arose from a strain which had circulated for several years in Kenya before recent detection, has experienced diversifying selection pressure, and may contain new putative immunogens relevant to vaccine design. These findings prompt further genomic epidemiology studies in this and other Kenyan locations to further elucidate the transmission dynamics of DENV in this region.Author summary (non-technical)Kenya experiences a substantial burden of dengue, yet the patterns of dengue spread in this region are unclear. Evolutionary analyses of dengue virus strain sequences could offer major insights into the spread of dengue viruses in this region, but there are very few DENV-2 sequence data available from this country and indeed the entire continent of Africa. We therefore undertook whole genome sequencing and evolutionary analysis of fourteen dengue virus (DENV)-2 strains sampled from Malindi sub-County Hospital during the 2017 DENV-2 outbreak in the Kenyan coast. We further performed an extended East African phylogenetic analysis, which leveraged 26 complete Africanenvgenes. Our results indicated transmission of DENV-2 viruses between East Africa and South/South-West Asia, that there is localized spread of DENV-2 viruses within Kenya, and that Kenyan DENV-2 strains were circulating for up to 5.38 years in Kenya before detection in the 2017 Coastal outbreak. We further performed selection pressure analyses and identified possible new markers of DENV-2 immune recognition specific to this population, with relevance to vaccine design.

BackgroundIn 2016, a chikungunya virus (CHIKV) outbreak was reported in Mandera, Kenya. This was the first major CHIKV outbreak in the country since the global re-emergence of this virus, which arose as an initial outbreak in Kenya in 2004. Therefore, we collected samples and sequenced viral genomes from the 2016 Mandera outbreak.Methodology/Principal FindingsAll Kenyan genomes contained two mutations, E1:K211E and E2:V264A, recently reported to have an association with increased infectivity, dissemination and transmission in the Aedes aegypti (Ae. aegypti) vector. Phylogeographic inference of temporal and spatial virus relationships using Bayesian approaches showed that this Ae. aegypti adapted strain emerged within the East, Central, and South African (ECSA) lineage of CHIKV between 2005 and 2008, most probably in India. It was also in India where the first large outbreak caused by this strain appeared, in New Delhi, 2010. More importantly, our results also showed that this strain is no longer contained to India, and that it has more recently caused several major outbreaks of CHIKV, including the 2016 outbreaks in India, Pakistan and Kenya, and the 2017 outbreak in Bangladesh. In addition to its capability to cause large outbreaks in different regions of the world, this CHIKV strain has the capacity to replace less adapted wild type strains in Ae. aegypti-rich regions. Indeed, all the latest full CHIKV genomes of the ECSA Indian Ocean Lineage (IOL), from the regions of high Ae. aegypti prevalence, carry these two mutations, including samples collected in Japan, Australia, and China.Conclusions/SignificanceOur results point to the importance of continued genomic-based surveillance of this strain’s global spread, and they prompt urgent vector competence studies in Asian and African countries, in order to assess the level of vector receptiveness, virus transmission, and the impact this might have on this strain’s ability to cause major outbreaks.Author summaryChikungunya virus (CHIKV) causes a debilitating infection with high fever, intense muscle and bone pain, rash, nausea, vomiting and headaches, and persistent and/or recurrent joint pains for months or years after contracting the virus. CHIKV is spread by two mosquito vectors, Aedes albopictus and Aedes aegypti, with increased presence around the globe. In this study, we report global spread of a CHIKV strain that carries two mutations that have been suggested to increase this virus’ ability to infect the Aedes aegypti mosquito, as well as to increase CHIKV’s ability to be transmitted by this vector. We show that this strain appeared sometime between 2005 and 2008, most probably in India, and has now spread to Africa, Asia, and Australia. We show that this strain is capable of driving large outbreaks of CHIKV in the human population, causing recent major outbreaks in Kenya, Pakistan, India and Bangladesh. Thus, our results stress the importance of monitoring this strain’s global spread, as well as the need of improved vector control strategies in the areas of Aedes aegypti prevalence.

Zika virus (ZIKV) is a flavivirus mainly transmitted to humans through the bite of infected Aedes aegypti mosquitoes. First isolated in Uganda in 1947, ZIKV was shown to circulate in enzootic sylvatic cycles in Africa and Asia for at least half a century before the first reported human epidemic occurred in 2007 on the Pacific island of Yap, Micronesia. Subsequently, larger ZIKV outbreaks were recorded in French Polynesia and other South Pacific islands during 2013-2014. In 2015, ZIKV reached Brazil from where it rapidly spread across the Americas and the Caribbean, causing hundreds of thousands of human cases. The factors that have fueled the explosiveness and magnitude of ZIKV emergence in the Pacific and the Americas are poorly understood. Reciprocally, the lack of major human epidemics of ZIKV in regions with seemingly favorable conditions, such as Africa or Asia, remains largely unexplained. To evaluate the potential contribution of vector population diversity to ZIKV epidemiological patterns, we established dose-response curves for eight field-derived Ae. aegypti populations representing the global range of the species, following experimental exposure to six low-passage ZIKV strains spanning the current viral genetic diversity. Our results reveal that African Ae. aegypti are significantly less susceptible than non-African Ae. aegypti across all ZIKV strains tested. We suggest that low susceptibility of vector populations may have contributed to prevent large-scale human transmission of ZIKV in Africa.