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Abstract Background Chemotherapy is a primary treatment for cancer, but its efficacy is often limited by cancer-associated bacteria (CAB) that impair tumor suppressor functions. Our previous research found that Mycoplasma fermentans DnaK, a chaperone protein, impairs p53 activities, which are essential for most anti-cancer chemotherapeutic responses. Methods To investigate the role of DnaK in chemotherapy, we treated cancer cell lines with M. fermentans DnaK and then with commonly used p53-dependent anti-cancer drugs (cisplatin and 5FU). We evaluated the cells’ survival in the presence or absence of a DnaK-binding peptide (ARV-1502). We also validated our findings using primary tumor cells from a novel DnaK knock-in mouse model. To provide a broader context for the clinical significance of these findings, we investigated human primary cancer sequencing datasets from The Cancer Genome Atlas (TCGA). We identified F. nucleatum as a CAB carrying DnaK with an amino acid composition highly similar to M. fermentans DnaK. Therefore, we investigated the effect of F. nucleatum DnaK on the anti-cancer activity of cisplatin and 5FU. Results Our results show that both M. fermentans and F. nucleatum DnaKs reduce the effectiveness of cisplatin and 5FU. However, the use of ARV-1502 effectively restored the drugs' anti-cancer efficacy. Conclusions Our findings offer a practical framework for designing and implementing novel personalized anti-cancer strategies by targeting specific bacterial DnaKs in patients with poor response to chemotherapy, underscoring the potential for microbiome-based personalized cancer therapies.

In the midst of an outbreak or sustained epidemic, reliable prediction of transmission risks and patterns of spread is critical to inform public health programs. Projections of transmission growth or decline among specific risk groups can aid in optimizing interventions, particularly when resources are limited. Phylogenetic trees have been widely used in the detection of transmission chains and high-risk populations. Moreover, tree topology and the incorporation of population parameters (phylodynamics) can be useful in reconstructing the evolutionary dynamics of an epidemic across space and time among individuals. We now demonstrate the utility of phylodynamic trees for transmission modeling and forecasting, developing a phylogeny-based deep learning system, referred to as DeepDynaForecast. Our approach leverages a primal-dual graph learning structure with shortcut multi-layer aggregation, which is suited for the early identification and prediction of transmission dynamics in emerging high-risk groups. We demonstrate the accuracy of DeepDynaForecast using simulated outbreak data and the utility of the learned model using empirical, large-scale data from the human immunodeficiency virus epidemic in Florida between 2012 and 2020. Our framework is available as open-source software (MIT license) at github.com/lab-smile/DeepDynaForcast.

The 2010 cholera epidemic in Haiti was thought to have ended in 2019, and the Prime Minister of Haiti declared the country cholera-free in February 2022. On September 25, 2022, cholera cases were again identified in Port-au-Prince. We compared genomic data from 42 clinical Vibrio cholerae strains from 2022 with data from 327 other strains from Haiti and 1,824 strains collected worldwide. The 2022 isolates were homogeneous and closely related to clinical and environmental strains circulating in Haiti during 2012–2019. Bayesian hypothesis testing indicated that the 2022 clinical isolates shared their most recent common ancestor with an environmental lineage circulating in Haiti in July 2018. Our findings strongly suggest that toxigenic V. cholerae O1 can persist for years in aquatic environmental reservoirs and ignite new outbreaks. These results highlight the urgent need for improved public health infrastructure and possible periodic vaccination campaigns to maintain population immunity against V. cholerae.

Vibrio mimicus caused a seafood-associated outbreak in Florida, USA, in which 4 of 6 case-patients were hospitalized; 1 required intensive care for severe diarrhea. Strains were ctx-negative but carried genes for other virulence determinants (hemolysin, proteases, and types I–IV and VI secretion systems). Cholera toxin–negative bacterial strains can cause cholera-like disease.

Abstract Background Accurate case report data are essential to understand arbovirus dynamics, including spread and evolution of arboviruses such as Zika, dengue and chikungunya viruses. Giving the multi-country nature of arbovirus epidemics in the Americas, these data are not often accessible or are reported at different time scales (weekly, monthly) from different sources. Results We developed a publicly available and user-friendly database for arboviral case data in the Americas: ARCA. ARCA is a relational database that is hosted on the ARCA website. Users can interact with the database through the website by submitting queries through the website, which generates displays results and allows users to download these results in different, convenient file formats. Users can choose to view arboviral case data through a table which containscontaining the number of cases for a particular week, a plot, or through a map. Conclusion Our ARCA database is a useful tool for arboviral epidemiology research allowing for complex queries, data visualization, integration, and formatting.

Cholera causes substantial illness and death in Africa. We analyzed 24 toxigenic Vibrio cholerae O1 strains isolated in 2015–2017 from patients in the Great Lakes region of the Democratic Republic of the Congo. Strains originating in southern Asia appeared to be part of the T10 introduction event in eastern Africa. We identified 2 main strain lineages, most recently a lineage corresponding to sequence type 515, a V. cholerae cluster previously reported in the Lake Kivu region. In 41% of fecal samples from cholera patients, we also identified a novel ICP1 (Bangladesh cholera phage 1) bacteriophage, genetically distinct from ICP1 isolates previously detected in Asia. Bacteriophage resistance occurred in distinct clades along both internal and external branches of the cholera phylogeny. This bacteriophage appears to have served as a major driver for cholera evolution and spread, and its appearance highlights the complex evolutionary dynamic that occurs between predatory phage and bacterial host.

BackgroundIn the wake of the SARS-CoV-2 pandemic, scientists have scrambled to collect and analyze SARS-CoV-2 genomic data to inform public health responses to COVID-19 in real time. Open source phylogenetic and data visualization platforms for monitoring SARS-CoV-2 genomic epidemiology have rapidly gained popularity for their ability to illuminate spatial-temporal transmission patterns worldwide. However, the utility of such tools to inform public health decision-making for COVID-19 in real time remains to be explored. ObjectiveThe aim of this study is to convene experts in public health, infectious diseases, virology, and bioinformatics—many of whom were actively engaged in the COVID-19 response—to discuss and report on the application of phylodynamic tools to inform pandemic responses. MethodsIn total, 4 focus groups (FGs) occurred between June 2020 and June 2021, covering both the pre- and postvariant strain emergence and vaccination eras of the ongoing COVID-19 crisis. Participants included national and international academic and government researchers, clinicians, public health practitioners, and other stakeholders recruited through purposive and convenience sampling by the study team. Open-ended questions were developed to prompt discussion. FGs I and II concentrated on phylodynamics for the public health practitioner, while FGs III and IV discussed the methodological nuances of phylodynamic inference. Two FGs per topic area to increase data saturation. An iterative, thematic qualitative framework was used for data analysis. ResultsWe invited 41 experts to the FGs, and 23 (56%) agreed to participate. Across all the FG sessions, 15 (65%) of the participants were female, 17 (74%) were White, and 5 (22%) were Black. Participants were described as molecular epidemiologists (MEs; n=9, 39%), clinician-researchers (n=3, 13%), infectious disease experts (IDs; n=4, 17%), and public health professionals at the local (PHs; n=4, 17%), state (n=2, 9%), and federal (n=1, 4%) levels. They represented multiple countries in Europe, the United States, and the Caribbean. Nine major themes arose from the discussions: (1) translational/implementation science, (2) precision public health, (3) fundamental unknowns, (4) proper scientific communication, (5) methods of epidemiological investigation, (6) sampling bias, (7) interoperability standards, (8) academic/public health partnerships, and (9) resources. Collectively, participants felt that successful uptake of phylodynamic tools to inform the public health response relies on the strength of academic and public health partnerships. They called for interoperability standards in sequence data sharing, urged careful reporting to prevent misinterpretations, imagined that public health responses could be tailored to specific variants, and cited resource issues that would need to be addressed by policy makers in future outbreaks. ConclusionsThis study is the first to detail the viewpoints of public health practitioners and molecular epidemiology experts on the use of viral genomic data to inform the response to the COVID-19 pandemic. The data gathered during this study provide important information from experts to help streamline the functionality and use of phylodynamic tools for pandemic responses.

ABSTRACT Toxigenic Vibrio cholerae O1 serotype Ogawa was introduced involuntarily into Haiti in October 2010, and virtually all of the clinical strains isolated during the first 5 years of the epidemic were Ogawa. Inaba strains were identified intermittently prior to 2015, with diverse mutations resulting in a common phenotype. In 2015, the percentage of clinical infections due to the Inaba serotype began to rapidly increase, with Inaba supplanting Ogawa as the dominant serotype during the subsequent 4 years. We investigated the molecular basis of the serotype switch and confirmed that all Inaba strains had the same level of mRNA expression of the wbeT genes, as well as the same translation levels for the truncated WbeT proteins in the V. cholerae Inaba isolates. Neither wbeT gene expression levels, differential mutations, or truncation size of the WbeT proteins appeared to be responsible for the successful Inaba switch in 2015. Our phylodynamic analysis demonstrated that the V. cholerae Inaba strains in Haiti evolved directly from Ogawa strains and that a significant increase of diversifying selection at the population level occurred at the time of the Ogawa-Inaba switch. We conclude that the emergence of the Inaba serotype was driven by diversifying selection, independent of the mutational pattern in the wbeT gene. IMPORTANCE Our phylodynamic analysis demonstrated that Vibrio cholerae Inaba strains in Haiti evolved directly from Ogawa strains. Our results support the hypothesis that after an initial Ogawa-dominated epidemic wave, V. cholerae Inaba was able to become the dominant strain thanks to a selective advantage driven by ongoing diversifying selection, independently from the mutational pattern in the wbeT gene.

ABSTRACT Florida is considered an epicenter of HIV in the United States. The U.S. federal plan for Ending the HIV Epidemic (EHE) within 10 years prioritizes seven of Florida’s 67 counties for intervention. We applied molecular epidemiology methods to characterize the HIV infection networks in the state and infer whether the results support the EHE. HIV sequences (N = 34,446) and associated clinical/demographic metadata of diagnosed people with HIV (PWH), during 2007 to 2017, were retrieved from the Florida Department of Health. HIV genetic networks were investigated using MicrobeTrace. Associates of clustering were identified through boosted logistic regression. Assortative trait mixing was also assessed. Bayesian phylogeographic methods were applied to evaluate evidence of imported HIV-1 lineages and illustrate spatiotemporal flows within Florida. We identified nine large clusters spanning all seven EHE counties but little evidence of external introductions, suggesting—in the absence of undersampling—an epidemic that evolved independently from the rest of the country or other external influences. Clusters were highly assortative by geography. Most of the sampled infections (82%) did not cluster with others in the state using standard molecular surveillance methods despite satisfactory sequence sampling in the state. The odds of being unclustered were higher among PWH in rural regions, and depending on demographics. A significant number of unclustered sequences were observed in counties omitted from EHE. The large number of missing sequence links may impact timely detection of emerging transmission clusters and ultimately hinder the success of EHE in Florida. Molecular epidemiology may help better understand infection dynamics at the population level and underlying disparities in disease transmission among subpopulations; however, there is also a continuous need to conduct ethical discussions to avoid possible harm of advanced methodologies to vulnerable groups, especially in the context of HIV stigmatization. IMPORTANCE The large number of missing phylogenetic linkages in rural Florida counties and among women and Black persons with HIV may impact timely detection of ongoing and emerging transmission clusters and ultimately hinder the success of epidemic elimination goals in Florida.

Objectives The objective of this study is the implementation of an automatic procedure to weekly detect new SARS-CoV-2 variants and non-neutral variants (variants of concern (VOC) and variants of interest (VOI)).Methods We downloaded spike protein primary sequences from the public resource GISAID and we represented each sequence as k-mer counts. For each week since 1 July 2020, we evaluate if each sequence represents an anomaly based on a One Class support vector machine (SVM) classification algorithm trained on neutral protein sequences collected from February to June 2020.Results We assess the ability of the One Class classifier to detect known VOC and VOI, such as Alpha, Delta or Omicron, ahead of their official classification by health authorities. In median, the classifier predicts a non-neutral variant as outlier 10 weeks before the official date of designation as VOC/VOI.Discussion The identification of non-neutral variants during a pandemic usually relies on indicators available during time, such as changing population size of a variant. Automatic variant surveillance systems based on protein sequences can enhance the fast identification of variants of potential concern.Conclusion Machine learning, and in particular One Class SVM classification, can support the detection of potentially VOC/VOI variants during an evolving pandemics.

Giovanetti et al. examine SARS-CoV-2 transmission dynamics in Italy using phylodynamic analysis of viral genetic and epidemiological data. They present evidence to suggest that the efficacy of public health interventions is limited by the size and structure of epidemic reservoirs, which may influence vaccination programmes.

Summary: Background: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. Methods: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). Findings: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10–30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. Interpretation: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19.

The dynamic nature of the SIV population during disease progression in the SIV/macaque model of AIDS and the factors responsible for its behavior have not been documented, largely owing to the lack of sufficient spatial and temporal sampling of both viral and host data from SIV-infected animals. In this study, we detail Bayesian coalescent inference of the changing collective intra-host viral effective population size (Ne) from various tissues over the course of infection and its relationship with what we demonstrate is a continuously changing immune cell repertoire within the blood. Although the relative contribution of these factors varied among hosts and time points, the adaptive immune response best explained the overall periodic dynamic behavior of the effective virus population. Data exposing the nature of the relationship between the virus and immune cell populations revealed the plausibility of an eco-evolutionary mathematical model, which was able to mimic the large-scale oscillations in Ne through virus escape from relatively few, early immunodominant responses, followed by slower escape from several subdominant and weakened immune populations. The results of this study suggest that SIV diversity within the untreated host is governed by a predator-prey relationship, wherein differing phases of infection are the result of adaptation in response to varying immune responses. Previous investigations into viral population dynamics using sequence data have focused on single estimates of the effective viral population size (Ne) or point estimates over sparse sampling data to provide insight into the precise impact of immune selection on virus adaptive behavior. Herein, we describe the use of the coalescent phylogenetic frame- work to estimate the relative changes in Ne over time in order to quantify the relationship with empirical data on the dynamic immune composition of the host. This relationship has allowed us to expand on earlier simulations to build a predator-prey model that explains the deterministic behavior of the virus over the course of disease progression. We show that sequential viral adaptation can occur in response to phases of varying immune pressure, providing a broader picture of the viral response throughout the entire course of progression to AIDS.

We report the identification of two orthobunyaviruses, Melao virus (MELV) and Oropouche virus (OROV), in plasma specimens from Haitian children with acute febrile illness who presented during outbreaks caused by alpha- and flaviviruses in 2014. Heretofore not described as a human pathogen, MELV was isolated in cell culture from the plasma of five case patients. OROV RNA was detected in the plasma of an additional child, using an unbiased sequencing approach, with phylogenetic inference suggesting a close relationship with strains from Brazil. Abdominal pain was reported by four case patients with MELV infections, with lymphadenopathy noted in two cases. Our findings document the occurrence of these orthobunyaviruses within the Caribbean region and highlight the critical importance of surveillance with viral genome sequence analyses to identify outbreaks caused by these and other emerging viruses.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that is being recognized with increasing frequency in South America. As part of on-going surveillance of a school cohort in Haiti, we identified MAYV infections in 5 children across a 7-month time span, at two different school campuses. All had a history of fever, and three had headaches; none complained of arthralgias. On analysis of whole genome sequence data, three strains were genotype D, and two were genotype L; phylogenetic and molecular clock analysis was consistent with at least 3 independent introductions of the virus into Haiti, with ongoing transmission of a common genotype D strain in a single school. Our data highlight the clear potential for spread of the virus in the northern Caribbean and North America.

Abstract Clonal lineages of ESBL (Extended-Spectrum β-Lactamase)-producing E. coli belonging to sequence type 131 (ST131) have disseminated globally during the last 30 years, leading to an increased prevalence of resistance to fluoroquinolones and extended-spectrum cephalosporins in clinical isolates of E. coli. We aimed to study if Swedish ESBL-producing ST131 isolates originated from single or multiple introductions to the population by assessing the amount of genetic variation, on chromosomal and plasmid level, between Swedish and international E. coli ST131. Bayesian inference of Swedish E. coli ST131 isolates (n = 29), sequenced using PacBio RSII, together with an international ST131 dataset showed that the Swedish isolates were part of the international ST131 A, C1 and C2 clades. Highly conserved plasmids were identified in three clusters although they were separated by several years, which indicates a strong co-evolution between some ST131 lineages and specific plasmids. In conclusion, the tight clonal relationship observed within the ST131 clades, together with highly conserved plasmids, challenges investigation of strain transmission events. A combination of few SNPs on a genome-wide scale and an epidemiological temporospatial link, are needed to track the spread of the ST131 subclones.

SARS-CoV-2, the causative agent of COVID-19, emerged in late 2019. The highly contagious B.1.617.2 (Delta) variant of concern (VOC) was first identified in October 2020 in India and subsequently disseminated worldwide, later becoming the dominant lineage in the US. Understanding the local transmission dynamics of early SARS-CoV-2 introductions may inform actionable mitigation efforts during subsequent pandemic waves. Yet, despite considerable genomic analysis of SARS-CoV-2 in the US, several gaps remain. Here, we explore the early emergence of the Delta variant in Florida, US using phylogenetic analysis of representative Florida and globally sampled genomes. We find multiple independent introductions into Florida primarily from North America and Europe, with a minority originating from Asia. These introductions led to three distinct clades that demonstrated varying relative rates of transmission and possessed five distinct substitutions that were 3–21 times more prevalent in the Florida sample as compared to the global sample. Our results underscore the benefits of routine viral genomic surveillance to monitor epidemic spread and support the need for more comprehensive genomic epidemiology studies of emerging variants. In addition, we provide a model of epidemic spread of newly emerging VOCs that can inform future public health responses.

In February and March, 2020, environmental surface swab samples were collected from the handle of the main entry door of a major university building in Florida, as part of a pilot surveillance project screening for influenza. Samples were taken at the end of regular classroom hours, between the dates of February 1-5 and February 19-March 4, 2020. Influenza A(H1N1)pdm09 virus was isolated from the door handle on four of the 19 days sampled. Both SARS-CoV-2 and A(H1N1)pdm09 virus were detected in a sample collected on February 21, 2020. Based on sequence analysis, the Florida SARS-CoV-2 strain (designated UF-11) was identical to strains being identified in Washington state during the same time period, while the earliest similar sequences were sampled in China/Hubei between Dec 30th 2019 and Jan 5th 2020. The first human case of COVID-19 was not officially reported in Florida until March 1st. In an analysis of sequences from COVID-19 patients in this region of Florida, there was only limited evidence of subsequent dissemination of the UF-11 strain. Identical or highly similar strains, possibly related through a common transmission chain, were detected with increasing frequency in Washington state between end of February and beginning of March. Our data provide further documentation of the rapid early spread of SARS-CoV-2 and underscore the likelihood that closely related strains were cryptically circulating in multiple U.S. communities before the first "official" cases were recognized.

ABSTRACT The malaria parasite, Plasmodium falciparum, was introduced into Hispaniola and other regions of the Americas through the slave trade spanning the 16th through the 19th centuries. During this period, more than 12 million Africans were brought across the Atlantic to the Caribbean and other regions of the Americas. Since malaria is holoendemic in West Africa, a substantial percentage of these individuals carried the parasite. St. Domingue on Hispaniola, now modern-day Haiti, was a major port of disembarkation, and malaria is still actively transmitted there. We undertook a detailed study of the phylogenetics of the Haitian parasites and those from Colombia and Peru utilizing whole-genome sequencing. Principal-component and phylogenetic analyses, based upon single nucleotide polymorphisms (SNPs) in protein coding regions, indicate that, despite the potential for millions of introductions from Africa, the Haitian parasites share an ancestral relationship within a well-supported monophyletic clade with parasites from South America, while belonging to a distinct lineage. This result, in stark contrast to the historical record of parasite introductions, is best explained by a severe population bottleneck experienced by the parasites introduced into the Americas. Here, evidence is presented for targeted selection of rare African alleles in genes which are expressed in the mosquito stages of the parasite’s life cycle. These genetic markers support the hypothesis that the severe population bottleneck was caused by the required adaptation of the parasite to transmission by new definitive hosts among the Anopheles (Nyssorhynchus) spp. found in the Caribbean and South America. IMPORTANCE Historical data suggest that millions of P. falciparum parasite lineages were introduced into the Americas during the trans-Atlantic slave trade, which would suggest a paraphyletic origin of the extant isolates in the Western Hemisphere. Our analyses of whole-genome variants show that the American parasites belong to a well-supported monophyletic clade. We hypothesize that the required adaptation to American vectors created a severe bottleneck, reducing the effective introduction to a few lineages. In support of this hypothesis, we discovered genes expressed in the mosquito stages of the life cycle that have alleles with multiple, high-frequency or fixed, nonsynonymous mutations in the American populations which are rarely found in African isolates. These alleles appear to be in gene products critical for transmission through the anopheline vector. Thus, these results may inform efforts to develop novel transmission-blocking vaccines by identifying parasite proteins functionally interacting with the vector that are important for successful transmission. Further, to the best of our knowledge, these are the first whole-genome data available from Haitian P. falciparum isolates. Defining the genome of these parasites provides genetic markers useful for mapping parasite populations and monitoring parasite movements/introductions.

HIV-1 subtype C is the most abundant strain of HIV-1 infections worldwide and was found in the first known patients diagnosed with HIV/AIDS in Bulgaria in 1986. However, there is limited information on the molecular-epidemiological characteristics of this strain in the epidemic of the country. In this study, we analyze the evolutionary history of the introduction and dissemination of HIV-1 subtype C in Bulgaria using global phylogenetic analysis, Bayesian coalescent-based approach, and molecular clock methods. All available samples with HIV-1 subtype C from individuals diagnosed with HIV/AIDS between 1986 and 2017 were analyzed. Men and women were equally represented, and 24.3% of patients reported being infected abroad. The global phylogenetic analysis indicated multiple introductions of HIV-1 subtype C from various countries of the world. The reconstruction of a Bayesian time-scaled phylogenies showed that several Bulgarian strains segregated together in clusters, while others were intermixed in larger clades containing strains isolated from both European and non-European countries. The time-scale of HIV-1 subtype C introductions in Bulgaria demonstrates the early introduction of these viruses in the country. Our in-depth phylogenetic and phylogeographic analyses are compatible with a scenario of multiple early introductions in the country followed by limited local distribution in the subsequent years. HIV-1 subtype C was introduced in the early years of the epidemic, originating from different countries of the world. Due to the comprehensive measures for prevention and control in the early years of the epidemic in Bulgaria, HIV-1 subtype C was not widely disseminated among the general population of the country.

ABSTRACT The incidence of locally acquired dengue infections increased during the last decade in the United States, compelling a sustained research effort concerning the dengue mosquito vector, Aedes aegypti, and its microbiome, which has been shown to influence virus transmission success. We examined the “metavirome” of four populations of Aedes aegypti mosquitoes collected in 2016 to 2017 in Manatee County, FL. Unexpectedly, we discovered that dengue virus serotype 4 (DENV4) was circulating in these mosquito populations, representing the first documented case of such a phenomenon in the absence of a local DENV4 human case in this county over a 2-year period. We confirmed that all of the mosquito populations carried the same DENV4 strain, assembled its full genome, validated infection orthogonally by reverse transcriptase PCR, traced the virus origin, estimated the time period of its introduction to the Caribbean region, and explored the viral genetic signatures and mosquito-specific virome associations that potentially mediated DENV4 persistence in mosquitoes. We discuss the significance of prolonged maintenance of the DENV4 infections in A. aegypti that occurred in the absence of a DENV4 human index case in Manatee County with respect to the inability of current surveillance paradigms to detect mosquito vector infections prior to a potential local outbreak. IMPORTANCE Since 1999, dengue outbreaks in the continental United States involving local transmission have occurred only episodically and only in Florida and Texas. In Florida, these episodes appear to be coincident with increased introductions of dengue virus into the region through human travel and migration from countries where the disease is endemic. To date, the U.S. public health response to dengue outbreaks has been largely reactive, and implementation of comprehensive arbovirus surveillance in advance of predictable transmission seasons, which would enable proactive preventative efforts, remains unsupported. The significance of our finding is that it is the first documented report of DENV4 transmission to and maintenance within a local mosquito vector population in the continental United States in the absence of a human case during two consecutive years. Our data suggest that molecular surveillance of mosquito populations in high-risk, high-tourism areas of the United States may enable proactive, targeted vector control before potential arbovirus outbreaks.

Objective: To evaluate the evolution of the pathogen Mayaro virus, causing Mayaro fever (a mosquito-borne disease) and to perform selective pressure analysis and homology modelling. Methods: Nine different datasets were built, one for each protein (from protein C to non-structural protein 4) and the last one for the complete genome. Selective pressure and homology modelling analyses were applied. Results: Two main clades (A and B) were pointed in the maximum likelihood tree. The clade A included five Brazilian sequences sampled from 1955 to 2015. The Brazilian sequence sampled in 2014 significantly clustered with the Haitian sequence sampled in 2015. The clade B included the remaining 27 sequences sampled in the Central and Southern America from 1957 to 2013. Selective pressure analysis revealed several sites under episodic diversifying selection in envelope surface glycoprotein E1, non-structural protein 1 and non- structural protein 3 with a posterior probability P≤0.01. Homology modelling showed different sites modified by selective pressure and some protein-protein interaction sites at high interaction propensity. Conclusion: Maximum likelihood analysis confirmed the Mayaro virus previous circulation in Haiti and the successful spread to the Caribbean and USA. Selective pressure analysis revealed a strong presence of negatively selected sites, suggesting a probable purging of deleterious polymorphisms in functional genes. Homology model showed the position 31, under selective pressure, located in the edge of the ADP-ribose binding site predicting to possess a high potential of protein-protein interaction and suggesting the possible chance for a protective vaccine, thus preventing Mayaro virus urbanization as with Chikungunya virus.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is primarily responsible for coronavirus disease (COVID-19) and it is characterized by respiratory illness with fever and dyspnea. Severe vascular problems and several other manifestations, including neurological ones, have also been frequently reported, particularly in the great majority of “long hauler” patients. SARS-CoV-2 infects and replicates in lung epithelial cells, while dysfunction of endothelial and neuronal brain cells has been observed in the absence of productive infection. It has been shown that the Spike protein can interact with specific cellular receptors, supporting both viral entry and cellular dysfunction. It is thus clear that understanding how and when these receptors are regulated, as well as how much they are expressed would help in unveiling the multifaceted aspects of this disease. Here, we show that SH-SY5Y neuroblastoma cells express three important cellular surface molecules that interact with the Spike protein, namely ACE2, TMPRSS2, and NRP1. Their levels increase when cells are treated with retinoic acid (RA), a commonly used agent known to promote differentiation. This increase matched the higher levels of receptors observed on HUVEC (primary human umbilical vein endothelial cells). We also show by confocal imaging that replication-defective pseudoviruses carrying the SARS-CoV-2 Spike protein can infect differentiated and undifferentiated SH-SY5Y, and HUVEC cells, although with different efficiencies. Neuronal cells and endothelial cells are potential targets for SARS-CoV-2 infection and the interaction of the Spike viral protein with these cells may cause their dysregulation. Characterizing RNA and protein expression tempo, mode, and levels of different SARS-CoV-2 receptors on both cell subpopulations may have clinical relevance for the diagnosis and treatment of COVID-19-infected subjects, including long hauler patients with neurological manifestations.

Abstract Mayaro virus (MAYV), causative agent of Mayaro Fever, is an arbovirus transmitted by Haemagogus mosquitoes. Despite recent attention due to the identification of several cases in South and Central America and the Caribbean, limited information on MAYV evolution and epidemiology exists and represents a barrier to prevention of further spread. We present a thorough spatiotemporal evolutionary study of MAYV full-genome sequences collected over the last sixty years within South America and Haiti, revealing recent recombination events and adaptation to a broad host and vector range, including Aedes mosquito species. We employed a Bayesian phylogeography approach to characterize the emergence of recombinants in Brazil and Haiti and report evidence in favor of the putative role of human mobility in facilitating recombination among MAYV strains from geographically distinct regions. Spatiotemporal characteristics of recombination events and the emergence of this previously neglected virus in Haiti, a known hub for pathogen spread to the Americas, warrants close monitoring of MAYV infection in the immediate future.

Abstract Carbapenems resistant Enterobacteriaceae infections are increasing worldwide representing an emerging public health problem. The application of phylogenetic and phylodynamic analyses to bacterial whole genome sequencing (WGS) data have become essential in the epidemiological surveillance of multi-drug resistant nosocomial pathogens. Between January 2012 and February 2013, twenty-one multi-drug resistant K. pneumoniae strains, were collected from patients hospitalized among different wards of the University Hospital Campus Bio-Medico. Epidemiological contact tracing of patients and Bayesian phylogenetic analysis of bacterial WGS data were used to investigate the evolution and spatial dispersion of K. pneumoniae in support of hospital infection control. The epidemic curve of incident K. pneumoniae cases showed a bimodal distribution of cases with two peaks separated by 46 days between November 2012 and January 2013. The time-scaled phylogeny suggested that K. pneumoniae strains isolated during the study period may have been introduced into the hospital setting as early as 2007. Moreover, the phylogeny showed two different epidemic introductions in 2008 and 2009. Bayesian genomic epidemiology is a powerful tool that promises to improve the surveillance and control of multi-drug resistant pathogens in an effort to develop effective infection prevention in healthcare settings or constant strains reintroduction.

Abstract Background Phylodynamics, the study of the interaction between epidemiological and pathogen evolutionary processes within and among populations, was originally defined in the context of rapidly evolving viruses and used to characterize transmission dynamics. The concept of phylodynamics has evolved since the early 21st century, extending its reach to slower-evolving pathogens, including bacteria and fungi, and to the identification of influential factors in disease spread and pathogen population dynamics. Results The phylodynamic approach has now become a fundamental building block for the development of comparative phylogenetic tools capable of incorporating epidemiological surveillance data with molecular sequences into a single statistical framework. These innovative tools have greatly enhanced scientific investigations of the temporal and geographical origins, evolutionary history, and ecological risk factors associated with the growth and spread of viruses such as human immunodeficiency virus (HIV), Zika, and dengue and bacteria such as Methicillin-resistant Staphylococcus aureus. Conclusions Capitalizing on an extensive review of the literature, we discuss the evolution of the field of infectious disease epidemiology and recent accomplishments, highlighting the advancements in phylodynamics, as well as the challenges and limitations currently facing researchers studying emerging pathogen epidemics across the globe.

Madariaga virus (MADV), also known as South American eastern equine encephalitis virus, has been identified in animals and humans in South and Central America, but not previously in Hispaniola or the northern Caribbean. MADV was isolated from virus cultures of plasma from an 8-year-old child in a school cohort in the Gressier/Leogane region of Haiti, who was seen in April, 2015, with acute febrile illness (AFI). The virus was subsequently cultured from an additional seven AFI case patients from this same cohort in February, April, and May 2016. Symptoms most closely resembled those seen with confirmed dengue virus infection. Sequence data were available for four isolates: all were within the same clade, with phylogenetic and molecular clock data suggesting recent introduction of the virus into Haiti from Panama sometime in the period from October 2012-January 2015. Our data document the movement of MADV into Haiti, and raise questions about the potential for further spread in the Caribbean or North America.

West Nile virus (WNV), a small, positive sense, single stranded RNA virus continues to encroach into new locales with emergence of new viral variants. Neurological disease in the equine can be moderate to severe in the face of low to undetectable virus loads. Physical methods of virus enrichment may increase sensitivity of virus detection and enhance analysis of viral diversity, especially for deep sequencing studies. However, the use of these techniques is limited mainly to non-neural tissues. We investigated the hypothesis that elimination of equine brain RNA enhances viral detection without limiting viral variation. Eight different WNV viral RNA enrichment and host RNA separation methods were evaluated to determine if elimination of host RNA enhanced detection of WNV and increase the repertoire of virus variants for sequencing. Archived brain tissue from 21 different horses was inoculated with WNV, homogenized, before enrichment and separation. The protocols utilized combinations of low-speed centrifugation, syringe filtration, and nuclease treatment. Viral and host RNA were analyzed using real-time PCR targeting the WNV Envelope (E) protein and equine G3PDH to determine relative sensitivity for WNV and host depletion, respectively. To determine the effect of these methods on viral variation, deep sequencing of the E protein was performed. Our results demonstrate that additional separation and enrichment methods resulted in loss of virus in the face of host RNA depletion. DNA sequencing showed no significant difference in total sequence variation between the RNA enrichment protocols. For equine brain infected with WNV, direct RNA extraction followed by host RNA depletion was most suitable. This study highlights the importance of evaluating viral enrichment and separation methods according to tissue type before embarking on studies where quantification of virus and viral variants is essential to the outcome of the study.

Our evolutionary and structural analyses revealed that the severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) spike gene is a complex mosaic resulting from several recombination events. Additionally, the fixation of variants has mainly been driven by purifying selection, suggesting the presence of conserved structural features. Our dynamic simulations identified two main long-range covariant dynamic movements of the novel glycoprotein, and showed that, as a result of the evolutionary duality, they are preserved. The first movement involves the receptor binding domain with the N-terminal domain and the C-terminal domain 2 and is maintained across human, bat and pangolin coronaviruses. The second is a complex network of long-range dynamics specific to SARS-CoV-2 involving the novel PRRA and the conserved KR*SF cleavage sites, as well as conserved segments in C-terminal domain 3. These movements, essential for host cell binding, are maintained by hinges conserved across human, bat, and pangolin coronaviruses glycoproteins. The hinges, located around Threonine 333 and Proline 527 within the N-terminal domain and C-terminal domain 2, represent candidate targets for the future development of novel pan-coronavirus inhibitors. In summary, we show that while recombination created a new configuration that increased the covariant dynamic movements of the SARS-CoV-2 glycoprotein, negative selection preserved its inter-domain structure throughout evolution in different hosts and inter-species transmissions.

Molecular HIV surveillance is a promising public health strategy for curbing the HIV epidemic. Clustering technologies used by health departments to date are limited in their ability to infer/forecast cluster growth trajectories. Resolution of the spatiotemporal dynamics of clusters, through phylodynamic and phylogeographic modelling, is one potential strategy to develop a forecasting tool; however, the projected utility of this approach needs assessment. Prior to incorporating novel phylodynamic-based molecular surveillance tools, we sought to identify possible issues related to their feasibility, acceptability, interpretation, and utility. Qualitative data were collected via focus groups among field experts (n = 17, 52.9% female) using semi-structured, open-ended questions. Data were coded using an iterative process, first through the development of provisional themes and subthemes, followed by independent line-by-line coding by two coders. Most participants routinely used molecular methods for HIV surveillance. All agreed that linking molecular sequences to epidemiological data is important for improving HIV surveillance. We found that, in addition to methodological challenges, a variety of implementation barriers are expected in relation to the uptake of phylodynamic methods for HIV surveillance. The participants identified several opportunities to enhance current methods, as well as increase the usability and utility of promising works-in-progress.

Mayaro virus has been associated with small outbreaks in northern South America. We isolated this virus from a child with acute febrile illness in rural Haiti, confirming its role as a cause of mosquitoborne illness in the Caribbean region. The clinical presentation can mimic that of chikungunya, dengue, and Zika virus infections.

Rotavirus remains a major cause of diarrhea in infants and young children worldwide. The permanent emergence of new genotypes puts the potential effectiveness of vaccines under serious question. The distribution of unusual genotypes subject to viral fitness is influenced by interactions among viral proteins. The present work aimed at analyzing the genetic constellation and the coevolution of rotavirus coding genes for the available rotavirus genotypes. Seventy-two full genome sequences of different genetic constellations were analyzed using a genetic algorithm. The results revealed an extensive genome-wide covariance network among the 12 viral proteins. Altogether, the emergence of new genotypes represents a challenge to the outcome and success of vaccination and the coevolutionary analysis of rotavirus proteins may boost efforts to better understand the interaction networks of proteins during viral replication/transcription.

In the context of recent arbovirus epidemics, questions about the frequency of simultaneous infection of patients with different arbovirus species have been raised. In 2014, a major Chikungunya virus (CHIKV) epidemic impacted the Caribbean and South America. As part of ongoing screening of schoolchildren presenting with acute undifferentiated febrile illness in rural Haiti, we used RT-PCR to identify CHIKV infections in 82 of 100 children with this diagnosis during May-August 2014. Among these, eight were infected with a second arbovirus: six with Zika virus (ZIKV), one with Dengue virus serotype 2, and one with Mayaro virus (MAYV). These dual infections were only detected following culture of the specimen, suggesting low viral loads of the co-infecting species. Phylogenetic analyses indicated that the ZIKV and MAYV strains differ from those detected later in 2014 and 2015, respectively. Moreover, CHIKV and ZIKV strains from co-infected patients clustered monophyletically in their respective phylogeny, and clock calibration traced back the common ancestor of each clade to an overlapping timeframe of introduction of these arboviruses onto the island.

As part of on-going arboviral surveillance activity in a semi-rural region in Haiti, Chikungunya virus (CHIKV)-positive mosquito pools were identified in 2014 (the peak of the Caribbean Asian-clade epidemic), and again in 2016 by RT-PCR. In 2014, CHIKV was only identified in Aedes aegypti (11 positive pools/124 screened). In contrast, in sampling in 2016, CHIKV was not identified in Ae. aegypti, but, rather, in (a) a female Aedes albopictus pool, and (b) a female Culex quinquefasciatus pool. Genomic sequence analyses indicated that the CHIKV viruses in the 2016 mosquito pools were from the East-Central-South African (ECSA) lineage, rather than the Asian lineage. In phylogenetic studies, these ECSA lineage strains form a new ECSA subgroup (subgroup IIa) together with Brazilian ECSA lineage strains from an isolated human outbreak in 2014, and a mosquito pool in 2016. Additional analyses date the most recent common ancestor of the ECSA IIa subgroup around May 2007, and the 2016 Haitian CHIKV genomes around December 2015. Known CHIKV mutations associated with improved Ae. albopictus vector competence were not identified. Isolation of this newly identified lineage from Ae. albopictus is of concern, as this vector has a broader geographic range than Ae. aegypti, especially in temperate areas of North America, and stresses the importance for continued vector surveillance.

The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death (Lamers et al., 2016a and 2016b; Rose et al., 2016). This method optimizes protocols from previous publications (Palmer et al., 2005; Norström et al., 2012; Lamers et al., 2015; 2016a and 2016b; Rife et al., 2016) to produce single distinct PCR products that can be directly sequenced and includes several cost-saving and time-efficient modifications.

During the past ten years, an increasing number of arbovirus outbreaks have affected tropical islands worldwide. We examined the available literature in peer-reviewed journals, from the second half of the 20th century until 2018, with the aim of gathering an overall picture of the emergence of arboviruses in these islands. In addition, we included information on environmental and social drivers specific to island setting that can facilitate the emergence of outbreaks. Within the context of the One Health approach, our review highlights how the emergence of arboviruses in tropical islands is linked to the complex interplay between their unique ecological settings and to the recent changes in local and global sociodemographic patterns. We also advocate for greater coordination between stakeholders in developing novel prevention and mitigation approaches for an intractable problem.

BACKGROUND:Zika virus (ZIKV), first isolated in Uganda in 1947, is currently spreading rapidly through South America and the Caribbean. In Brazil, infection has been linked with microcephaly and other serious complications, leading to declaration of a public health emergency of international concern; however, there currently are only limited data on the virus (and its possible sources and manifestations) in the Caribbean. METHODS:From May, 2014-February, 2015, in conjunction with studies of chikungunya (CHIKV) and dengue (DENV) virus infections, blood samples were collected from children in the Gressier/Leogane region of Haiti who presented to a school clinic with undifferentiated febrile illness. Samples were initially screened by RT-PCR for CHIKV and DENV, with samples negative in these assays further screened by viral culture. FINDINGS:Of 177 samples screened, three were positive for ZIKV, confirmed by viral sequencing; DENV-1 was also identified in culture from one of the three positive case patients. Patients were from two different schools and 3 different towns, with all three cases occurring within a single week, consistent with the occurrence of an outbreak in the region. Phylogenetic analysis of known full genome viral sequences demonstrated a close relationship with ZIKV from Brazil; additional analysis of the NS5 gene, for which more sequences are currently available, showed the Haitian strains clustering within a monophyletic clade distinct from Brazilian, Puerto Rican and Guatemalan sequences, with all part of a larger clade including isolates from Easter Island. Phylogeography also clarified that at least three major African sub-lineages exist, and confirmed that the South American epidemic is most likely to have originated from an initial ZIKV introduction from French Polynesia into Easter Island, and then to the remainder of the Americas. CONCLUSIONS:ZIKV epidemics in South America, as well as in Africa, show complex dissemination patterns. The virus appears to have been circulating in Haiti prior to the first reported cases in Brazil. Factors contributing to transmission and the possible linkage of this early Haitian outbreak with microcephaly remain to be determined.

Despite infection prevention efforts, neonatal intensive care unit (NICU) patients remain at risk of Methicillin-resistant Staphylococcus aureus (MRSA) infection. Modes of transmission for healthcare-associated (HA) and community-associated (CA) MRSA remain poorly understood and may vary by genotype, hindering the development of effective prevention and control strategies. From 2008-2010, all patients admitted to a level III NICU were screened for MRSA colonization, and all available isolates were spa-typed. Spa-type t008, the most prevalent CA- genotype in the United States, spa-type t045, a HA- related genotype, and a convenience sample of strains isolated from 2003-2011, underwent whole-genome sequencing and phylodynamic analysis. Patient risk factors were compared between colonized and noncolonized infants, and virulence and resistance genes compared between spa-type t008 and non-t008 strains. Epidemiological and genomic data were used to estimate MRSA importations and acquisitions through transmission reconstruction. MRSA colonization was identified in 9.1% (177/1940) of hospitalized infants and associated with low gestational age and birth weight. Among colonized infants, low gestational age was more common among those colonized with t008 strains. Our data suggest that approximately 70% of colonizations were the result of transmission events within the NICU, with the remainder likely to reflect importations of "outside" strains. While risk of transmission within the NICU was not affected by spa-type, patterns of acquisition and importation differed between t008 and t045 strains. Phylodynamic analysis showed the effective population size of spa-type t008 has been exponentially increasing in both community and hospital, with spa-type t008 strains possessed virulence genes not found among t045 strains; t045 strains, in contrast, appeared to be of more recent origin, with a possible hospital source. Our data highlight the importance of both intra-NICU transmission and recurrent introductions in maintenance of MRSA colonization within the NICU environment, as well as spa-type-specific differences in epidemiology.

Kaposi’s sarcoma (KS) in HIV-infected individuals can have a wide range of clinical outcomes, from indolent skin tumors to a life-threatening visceral cancer. KS tumors contain endothelial-related cells and inflammatory cells that may be HIV-infected. In this study we tested if HIV evolutionary patterns distinguish KS tumor relatedness and progression. Multisite autopsies from participants who died from HIV-AIDS with KS prior to the availability of antiretroviral therapy were identified at the AIDS and Cancer Specimen Resource (ACSR). Two patients (KS1 and KS2) died predominantly from non-KS-associated disease and KS3 died due to aggressive and metastatic KS within one month of diagnosis. Skin and visceral tumor and nontumor autopsy tissues were obtained (n=12). Single genome sequencing was used to amplify HIV RNA and DNA, which was present in all tumors. Independent HIV tumor clades in phylogenies differentiated KS1 and KS2 from KS3, whose sequences were interrelated by both phylogeny and selection. HIV compartmentalization was confirmed in KS1 and KS2 tumors; however, in KS3, no compartmentalization was observed among sampled tissues. While the sample size is small, the HIV evolutionary patterns observed in all patients suggest an interplay between tumor cells and HIV-infected cells which provides a selective advantage and could promote KS progression.

ABSTRACT Phylodynamic analysis of genome-wide single-nucleotide polymorphism (SNP) data is a powerful tool to investigate underlying evolutionary processes of bacterial epidemics. The method was applied to investigate a collection of 65 clinical and environmental isolates of Vibrio cholerae from Haiti collected between 2010 and 2012. Characterization of isolates recovered from environmental samples identified a total of four toxigenic V. cholerae O1 isolates, four non-O1/O139 isolates, and a novel nontoxigenic V. cholerae O1 isolate with the classical tcpA gene. Phylogenies of strains were inferred from genome-wide SNPs using coalescent-based demographic models within a Bayesian framework. A close phylogenetic relationship between clinical and environmental toxigenic V. cholerae O1 strains was observed. As cholera spread throughout Haiti between October 2010 and August 2012, the population size initially increased and then fluctuated over time. Selection analysis along internal branches of the phylogeny showed a steady accumulation of synonymous substitutions and a progressive increase of nonsynonymous substitutions over time, suggesting diversification likely was driven by positive selection. Short-term accumulation of nonsynonymous substitutions driven by selection may have significant implications for virulence, transmission dynamics, and even vaccine efficacy. IMPORTANCE Cholera, a dehydrating diarrheal disease caused by toxigenic strains of the bacterium Vibrio cholerae, emerged in 2010 in Haiti, a country where there were no available records on cholera over the past 100 years. While devastating in terms of morbidity and mortality, the outbreak provided a unique opportunity to study the evolutionary dynamics of V. cholerae and its environmental presence. The present study expands on previous work and provides an in-depth phylodynamic analysis inferred from genome-wide single nucleotide polymorphisms of clinical and environmental strains from dispersed geographic settings in Haiti over a 2-year period. Our results indicate that even during such a short time scale, V. cholerae in Haiti has undergone evolution and diversification driven by positive selection, which may have implications for understanding the global clinical and epidemiological patterns of the disease. Furthermore, the continued presence of the epidemic strain in Haitian aquatic environments has implications for transmission.

Four SIV-infected monkeys with high plasma virus and CNS injury were treated with an anti-α4 blocking antibody (natalizumab) once a week for three weeks beginning on 28 days post-infection (late). Infection in the brain and gut were quantified, and neuronal injury in the CNS was assessed by MR spectroscopy, and compared to controls with AIDS and SIV encephalitis. Treatment resulted in stabilization of ongoing neuronal injury (NAA/Cr by 1H MRS), and decreased numbers of monocytes/macrophages and productive infection (SIV p28+, RNA+) in brain and gut. Antibody treatment of six SIV infected monkeys at the time of infection (early) for 3 weeks blocked monocyte/macrophage traffic and infection in the CNS, and significantly decreased leukocyte traffic and infection in the gut. SIV - RNA and p28 was absent in the CNS and the gut. SIV DNA was undetectable in brains of five of six early treated macaques, but proviral DNA in guts of treated and control animals was equivalent. Early treated animals had low-to-no plasma LPS and sCD163. These results support the notion that monocyte/macrophage traffic late in infection drives neuronal injury and maintains CNS viral reservoirs and lesions. Leukocyte traffic early in infection seeds the CNS with virus and contributes to productive infection in the gut. Leukocyte traffic early contributes to gut pathology, bacterial translocation, and activation of innate immunity.

Bats are natural reservoirs of coronaviruses and other viruses with zoonotic potential. Florida has indigenous non-migratory populations of Brazilian free-tailed bats (Tadarida brasiliensis) that mostly roost in colonies in artificial structures. Unlike their counterparts in Brazil and Mexico, the viruses harbored by the Florida bats have been underexplored. We report the detection of an alphacoronavirus RNA-dependent RNA polymerase (RdRp) gene sequence in the feces of two of 19 different T. brasiliensis that were capture/release bats that had been evaluated for overall health. The RdRp sequence is similar but not identical to previously detected sequences in the feces of two different species of bats (T. brasiliensis and Molossus molossus) in Brazil. In common with the experience of others doing similar work, attempts to isolate the virus in cell cultures were unsuccessful. We surmise that this and highly related alphacoronavirus are carried by Brazilian free-tailed bats living in a wide eco-spatial region. As various coronaviruses (CoVs) that affect humans emerged from bats, our study raises the question whether CoVs such as the one detected in our work are yet-to-be-detected pathogens of humans and animals other than bats.

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although risk of progression may vary among patients carrying this allele. The interplay between HIV-1 evolutionary rate variation and risk of progression to AIDS in HLA-B*5701 subjects was studied using longitudinal viral sequences from high-risk progressors (HRPs) and low-risk progressors (LRPs). Posterior distributions of HIV-1 genealogies assuming a Bayesian relaxed molecular clock were used to estimate the absolute rates of nonsynonymous and synonymous substitutions for different set of branches. Rates of viral evolution, as well as in vitro viral replication capacity assessed using a novel phenotypic assay, were correlated with various clinical parameters. HIV-1 synonymous substitution rates were significantly lower in LRPs than HRPs, especially for sets of internal branches. The viral population infecting LRPs was also characterized by a slower increase in synonymous divergence over time. This pattern did not correlate to differences in viral fitness, as measured by in vitro replication capacity, nor could be explained by differences among subjects in T cell activation or selection pressure. Interestingly, a significant inverse correlation was found between baseline CD4+ T cell counts and mean HIV-1 synonymous rate (which is proportional to the viral replication rate) along branches representing viral lineages successfully propagating through time up to the last sampled time point. The observed lower replication rate in HLA-B*5701 subjects with higher baseline CD4+ T cell counts provides a potential model to explain differences in risk of disease progression among individuals carrying this allele.