Independent infections of porcine deltacoronavirus among Haitian children.

Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, the emergence of coronavirus in our species has been associated with zoonotic transmissions from animal reservoirs1,2, underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae, human infections reported so far have been limited to alphacoronaviruses and betacoronaviruses3-5. Here we identify porcine deltacoronavirus strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the genes encoding Nsp15 and the spike glycoprotein. In particular, structural analysis predicts that one of the changes in the spike S1 subunit, which contains the receptor-binding domain, may affect the flexibility of the protein and its binding to the host cell receptor. Our findings highlight the potential for evolutionary change and adaptation leading to human infections by coronaviruses outside of the previously recognized human-associated coronavirus groups, particularly in settings where there may be close human-animal contact.

Keystone Virus Isolated From a Florida Teenager With Rash and Subjective Fever: Another Endemic Arbovirus in the Southeastern United States?

Keystone virus, a California-serogroup orthobunyavirus, was first isolated in 1964 from mosquitoes in Keystone, Florida. There were no prior reports of isolation from humans, despite studies suggesting that ~20% of persons living in the region are seropositive. We report virus isolation from a Florida teenager with a rash and fever.

Alcohol retail sales licenses and sexually transmitted infections in Texas counties, 2008-2015.

BACKGROUND: Research suggests that reduced retail alcohol outlet density may be associated with lower prevalence of HIV and other sexually transmitted infections (STIs). On-premise sale of alcohol for immediate consumption is theorized as increasing social interactions that can lead to sexual encounters. OBJECTIVE: We examined associations between on- and off-premise retail alcohol sales licenses and number of newly diagnosed HIV and STI cases in Texas counties. METHODS: Retail alcohol sales license data were obtained from the Texas Alcoholic Beverage Commission. HIV and bacterial STI data were obtained from the Texas Department of State Health Services. Associations between retail alcohol sales licenses and STIs were estimated using spatial linear models and Poisson mixed effects models for over-dispersed count data. RESULTS: Adjusting for county-specific confounders, there was no evidence of residual spatial correlation. In Poisson models, each additional on-premise (e.g., bar and restaurant) alcohol license per 10,000 population in a county was associated with a 1.5% increase (95% CI: 0.4%, 2.6%) in the rate of HIV and a 2.4% increase (95% CI: 1.9%, 3.0%) in the rate of bacterial STIs, adjusting for potential confounders. In contrast, number of off-premise licenses (e.g., take-out stores) was inversely associated with the incidence of STI and HIV, although the association with HIV was not statistically significant. CONCLUSIONS: This study adds to the limited literature on the association between retail alcohol availability and STIs. Additional research is needed on the role of alcohol availability (and policies affecting availability) in the spread of HIV and other STIs.

Brazilian Jiu Jitsu, Judo, and Mixed Martial Arts Injuries Presenting to United States Emergency Departments, 2008-2015.

Limited research has systematically examined injuries resulting from Brazilian Jiu Jitsu (BJJ), Judo, and mixed martial arts (MMA), especially when compared to more well-known or -established martial arts. These three combative sports differ substantially regarding their rules and techniques. BJJ emphasizes ground positioning and submission, Judo rewards throwing an opponent on their back with submission ending the match, and MMA emphasizes knocking out or forcing the submission of one's opponent. We examined injuries, among people of any age, experienced from participation in BJJ, Judo, and MMA. We analyzed data from the United States (U.S.) Consumer Product Safety Commission National Electronic Injury Surveillance System to create estimates of injuries presenting to U.S. emergency departments (EDs). We compared injury profiles between sports, including estimated numbers of injuries, their site, type, and mechanism. Participation in BJJ, Judo, and MMA resulted in an estimated 39,181 injuries presenting to U.S. EDs from 2008 through 2015. Strains and sprains were the most common diagnoses for BJJ and Judo participants, whereas abrasions/contusions were the most commonly diagnosed MMA injury. Being struck resulted in the majority of injuries for all three sports. The head was the most injured body region for BJJ and MMA, whereas the leg was the most injured body region for Judo. Finally, the majority of BJJ and Judo injuries occurred during noncompetitive grappling, whereas most MMA injuries occurred during competition. Our study adds to the limited literature examining injuries from BJJ, Judo, and MMA using data from a probability sample and is an initial step towards understanding the national burden of injury from participation in these sports. Given the quantity and severity of injuries sustained by participants, additional research is needed to assess the riskiness of participation and the effectiveness of interventions, such as improved personal protective gear and mats, as a means to prevent commonly occurring injuries.

Factors Associated with Self-Estimated Breath Alcohol Concentration Among Bar Patrons.

BACKGROUND: Few studies have examined the context in which drinkers underestimate their breath alcohol concentration (BrAC) in natural drinking environments. This study examined factors associated with bar patrons' self-estimated BrAC in high-risk college town settings. METHODS: Guided interview and BrAC data were collected from 510 participants recruited as they exited bars located close to large universities: 1 in Florida and 1 in Texas. RESULTS: Participants with the highest measured BrACs underestimated their BrAC levels the most. Findings from multivariable linear regression analysis indicated that BrAC (std ß = 0.014, p < 0.001), number of alcoholic drinks consumed (std ß = 0.006, p < 0.01), and perceived drunkenness (std ß = 0.024, p < 0.001) had significant positive associations with BrAC self-estimates, where the regression coefficients were scaled by values approximately equal to each variable's interquartile range. Among the 321 participants with BrAC levels ≥ 0.08 g/dl, 21.2% believed their BrAC was below the legal per se driving limit of 0.08 g/dl. Results from a logistic regression analysis indicated that higher levels of perceived drunkenness were associated with better self-recognition that one's BrAC level exceeded the legal driving threshold (OR = 3.312, p < 0.001). Further, participants under 26 years of age had reduced odds of recognizing that their BrAC was greater than 0.079 g/dl (OR = 0.245, p < 0.05). CONCLUSIONS: These findings highlight the inaccuracy of self-estimated BrAC when drinking, particularly among younger drinkers. Adjusting for BrAC, situational factors were strongly associated with self-estimated BrAC. Future research is needed to better understand how altering drinking environments may improve accuracy of BrAC self-estimates and deter driving after drinking.

Parkour injuries presenting to United States emergency departments, 2009-2015.

BACKGROUND: Few studies have examined injuries resulting from practicing parkour. The current study provides details on more parkour-related emergency department visits than existed in the combined research literature. METHODS: Cross-sectional data from the U.S. Consumer Product Safety Commission's National Electronic Injury Surveillance System were used to examine parkour-related injuries presenting to U.S. emergency departments over a seven year period. RESULTS: Most parkour injuries were reportedly caused by landing or from striking objects. Common diagnoses included fractures, sprains/strains, abrasions/contusions, and lacerations. More than half of reported injuries (57.7%) affected the patients' extremities. In this data, there appears to be a trend of increasing parkour injuries over time. Patients as young as 8years old have presented to emergency departments with parkour-related injuries in recent years. CONCLUSIONS: Given the relatively severe injuries obtained by youth participants, more research is needed to guide injury prevention efforts. Understanding the nature of parkour-related injuries may help inform prevention efforts. However, more systematic monitoring of participation in parkour and related injuries is needed.

Alteration of lysosome fusion and low-grade inflammation mediated by super-low-dose endotoxin.

Subclinical super-low-dose endotoxin LPS is a risk factor for the establishment of low-grade inflammation during the pathogenesis and progression of chronic diseases. However, the underlying mechanisms are not well understood. At the cellular level, a disruption of lysosome fusion with endosomes or autophagosomes may contribute to the potentiation of low-grade inflammation. In this study, we identified that subclinical super-low-dose endotoxin LPS can potently inhibit the process of endosome acidification and lysosome fusion with endosomes or autophagosomes in primary macrophages. Super-low-dose LPS induced the inhibitory phosphorylation of VPS34, thus leading to the disruption of endosome-lysosome fusion. This effect may depend upon the clearance and relocation of Tollip in macrophages by super-low-dose LPS. Consistent with this notion, Tollip-deficient macrophages had constitutively elevated levels of VPS34 inhibitory phosphorylation and constitutive disruption of endosome-lysosome fusion. By employing a skin excision wound-healing model, we observed that Tollip-deficient mice had significantly elevated levels of cell stress and reduced wound repair. This study reveals a novel mechanism responsible for the modulation of endosome-lysosome fusion and low-grade inflammation in innate macrophages.

Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism.

Introduction: SARS-CoV-2 subverts host cell processes to facilitate rapid replication and dissemination, and this leads to pathological inflammation. Methods: We used niclosamide (NIC), a poorly soluble anti-helminth drug identified initially for repurposed treatment of COVID-19, which activates the cells' autophagic and lipophagic processes as a chemical probe to determine if it can modulate the host cell's total lipid profile that would otherwise be either amplified or reduced during SARS-CoV-2 infection. Results: Through parallel lipidomic and transcriptomic analyses we observed massive reorganization of lipid profiles of SARS-CoV-2 infected Vero E6 cells, especially with triglycerides, which were elevated early during virus replication, but decreased thereafter, as well as plasmalogens, which were elevated at later timepoints during virus replication, but were also elevated under normal cell growth. These findings suggested a complex interplay of lipid profile reorganization involving plasmalogen metabolism. We also observed that NIC treatment of both low and high viral loads does not affect virus entry. Instead, NIC treatment reduced the abundance of plasmalogens, diacylglycerides, and ceramides, which we found elevated during virus infection in the absence of NIC, resulting in a significant reduction in the production of infectious virions. Unexpectedly, at higher viral loads, NIC treatment also resulted in elevated triglyceride levels, and induced significant changes in phospholipid metabolism. Discussion: We posit that future screens of approved or new partner drugs should prioritize compounds that effectively counter SARS-CoV-2 subversion of lipid metabolism, thereby reducing virus replication, egress, and the subsequent regulation of key lipid mediators of pathological inflammation.

Imported Dengue Case Numbers and Local Climatic Patterns Are Associated with Dengue Virus Transmission in Florida, USA.

Aedes aegypti mosquitoes are the main vector of dengue viruses globally and are present throughout much of the state of Florida (FL) in the United States of America. However, local transmission of dengue viruses in FL has mainly occurred in the southernmost counties; specifically Monroe and Miami-Dade counties. To get a better understanding of the ecologic risk factors for dengue fever incidence throughout FL, we collected and analyzed numerous environmental factors that have previously been connected to local dengue cases in disease-endemic regions. We analyzed these factors for each county-year in FL, between 2009-2019, using negative binomial regression. Monthly minimum temperature of 17.5-20.8 °C, an average temperature of 26.1-26.7 °C, a maximum temperature of 33.6-34.7 °C, rainfall between 11.4-12.7 cm, and increasing numbers of imported dengue cases were associated with the highest risk of dengue incidence per county-year. To our knowledge, we have developed the first predictive model for dengue fever incidence in FL counties and our findings provide critical information about weather conditions that could increase the risk for dengue outbreaks as well as the important contribution of imported dengue cases to local establishment of the virus in Ae. aegypti populations.

Genome Sequence of Genotype 1A Hepatovirus A Isolated from Plasma from a Haitian Child.

Genotype 1A hepatovirus A was identified by quantitative reverse transcription-PCR and isolated from plasma from a Haitian child with acute undifferentiated febrile illness and malaise. The strain was most closely related to Brazilian strains, consistent with recognized patterns of virus movement in the Caribbean region.

Analysis of Zika Virus Sequence Data Associated with a School Cohort in Haiti.

Zika virus (ZIKV) infections occurred in epidemic form in the Americas in 2014-2016, with some of the earliest isolates in the region coming from Haiti. We isolated ZIKV from 20 children with acute undifferentiated febrile illness who were part of a cohort of children seen at a school clinic in the Gressier region of Haiti. The virus was also isolated from three pools of Aedes aegypti mosquitoes collected at the same location. On phylogenetic analysis, three distinct ZIKV clades were identified. Strains from all three clades were present in Haiti in 2014, making them among the earliest isolates identified in the Western Hemisphere. Strains from all three clades were also isolated in 2016, indicative of their persistence across the time period of the epidemic. Mosquito isolates were collected in 2016 and included representatives from two of the three clades; in one instance, ZIKV was isolated from a pool of male mosquitoes, suggestive of vertical transmission of the virus. The identification of multiple ZIKV clades in Haiti at the beginning of the epidemic suggests that Haiti served as a nidus for transmission within the Caribbean.

A molecular surveillance-guided vector control response to concurrent dengue and West Nile virus outbreaks in a COVID-19 hotspot of Florida.

Background: Simultaneous dengue virus (DENV) and West Nile virus (WNV) outbreaks in Florida, USA, in 2020 resulted in 71 dengue virus serotype 1 and 86 WNV human cases. We hypothesized that we would find a number of DENV-1 positive mosquito pools, and that the distribution of these arbovirus-positive mosquito pools would be associated with those neighborhoods for which imported DENV cases have been recently reported in 2019 and 2020. Methods: We collected and screened Aedes aegypti, Ae. albopictus, Anopheles crucians, Culex coronator, Cx. nigripalpus, and Cx. quinquefasciatus mosquitoes from Miami-Dade County (Florida) for DENV and WNV by rRT-qPCR. Spatial statistical analyses were performed to capture positive mosquito pool distribution in relation to land use, human demography, environmental variables, mosquito trap placement and reported human travel associated DENV cases to guide future mosquito control outbreak responses. Findings: A rapid screen of 7,668 mosquitoes detected four DENV serotype 2 (DENV-2), nine DENV-4 and nine WNV-positive mosquito pools, which enabled swift and targeted abatement of trap sites by mosquito control. As expected, DENV-positive pools were in urban areas; however, we found WNV-positive mosquito pools in agricultural and recreational areas with no historical reports of WNV transmission. Interpretation: These findings demonstrate the importance of proactive arbovirus surveillance in mosquito populations to prevent and control outbreaks, particularly when other illnesses (e.g., COVID-19), which present with similar symptoms, are circulating concurrently. Growing evidence for substantial infection prevalence of dengue in mosquitoes in the absence of local index cases suggests a higher level of dengue endemicity in Florida than previously thought. Funding: This research was supported in part by U.S. Centers for Disease Control and Prevention (CDC) grant 1U01CK000510-03, Southeastern Regional Center of Excellence in Vector Borne Diseases Gateway Program.

Transmission Potential of Floridian Aedes aegypti Mosquitoes for Dengue Virus Serotype 4: Implications for Estimating Local Dengue Risk.

Dengue virus serotype 4 (DENV-4) circulated in Aedes aegypti in 2016 and 2017 in Florida in the absence of human index cases, compelling a full assessment of local mosquito vector competence and DENV-4 risk. To better understand DENV-4 transmission risk in Florida, we used an expanded suite of tests to measure and compare the vector competencies of both an established colony of A. aegypti (Orlando strain [ORL]) and a field-derived colony from Collier County, FL, in 2018 (COL) for a Haitian DENV-4 human field isolate and a DENV-4 laboratory strain (Philippines H241). We immediately noted that ORL saliva positivity was higher for the field than for laboratory DENV-4 strains. In a subsequent comparison with the recent COL mosquito colony, we also observed significantly higher midgut infection of COL and ORL by the Haitian DENV-4 field strain and a significantly higher saliva positivity rate for COL, although overall saliva virus titers were similar between the two. These data point to a potential midgut infection barrier for the DENV-4 laboratory strain for both mosquito colonies and indicate that the marked differences in transmission potential estimates hinge on virus-vector combinations. Our study highlights the importance of leveraging an expanded suite of testing methods with emphasis on utilizing local mosquito populations and field-relevant dengue virus serotypes and strains to accurately estimate transmission risk in a given setting. IMPORTANCE DENV-4 was found circulating in Florida (FL) A. aegypti mosquitoes in the absence of human index cases in the state (2016 to 2017). How DENV-4 was maintained locally is unclear, presenting a major gap in our understanding of DENV-4 public health risk. We determined the baseline arbovirus transmission potential of laboratory and field colonies of A. aegypti for both laboratory and field isolates of DENV-4. We observed a high transmission potential of field populations of A. aegypti and evidence of higher vertical transmission of the DENV-4 field isolate, providing clues to the possible mechanism of undetected DENV-4 maintenance in the state. Our findings also move the field forward in the development of best practices for evaluating arbovirus vector competence, with evidence that transmission potential estimates vary depending on the mosquito-virus combinations. These data emphasize the poor suitability of laboratory-established virus strains and the high relevance of field-derived mosquito populations in estimating transmission risk.

Infection Kinetics and Transmissibility of a Reanimated Dengue Virus Serotype 4 Identified Originally in Wild Aedes aegypti From Florida.

Dengue virus is the most prevalent mosquito-borne virus, causing approximately 390 million infections and 25,000 deaths per year. Aedes aegypti, the primary mosquito vector of dengue virus, is well-established throughout the state of Florida, United States. Autochthonous transmission of dengue virus to humans in Florida has been increasing since 2009, alongside consistent importation of dengue cases. However, most cases of first infection with dengue are asymptomatic and the virus can be maintained in mosquito populations, complicating surveillance and leading to an underestimation of disease risk. Metagenomic sequencing of A. aegypti mosquitoes in Manatee County, Florida revealed the presence of dengue virus serotype 4 (DENV-4) genomes in mosquitoes from multiple trapping sites over 2years, in the absence of a human DENV-4 index case, and even though a locally acquired case of DENV-4 has never been reported in Florida. This finding suggested that: (i) DENV-4 may circulate among humans undetected; (ii) the virus was being maintained in the mosquito population, or (iii) the detected complete genome sequence may not represent a viable virus. This study demonstrates that an infectious clone generated from the Manatee County DENV-4 (DENV-4M) sequence is capable of infecting mammalian and insect tissue culture systems, as well as adult female A. aegypti mosquitoes when fed in a blood meal. However, the virus is subject to a dose dependent infection barrier in mosquitoes, and has a kinetic delay compared to a phylogenetically related wild-type (WT) control virus from a symptomatic child, DENV-4H (strain Homo sapiens/Haiti-0075/2015, GenBank accession MK514144.1). DENV-4M disseminates from the midgut to the ovary and saliva at 14days post-infection. Viral RNA was also detectable in the adult female offspring of DENV-4M infected mosquitoes. These results demonstrate that the virus is capable of infecting vector mosquitoes, is transmissible by bite, and is vertically transmitted, indicating a mechanism for maintenance in the environment without human-mosquito transmission. These findings suggest undetected human-mosquito transmission and/or long-term maintenance of the virus in the mosquito population is occurring in Florida, and underscore the importance of proactive surveillance for viruses in mosquitoes. GRAPHICAL ABSTRACTIn order to better assess the public health risk posed by a detection of DENV-4 RNA in Manatee County, FL Aedes aegypti, we produced an infectious clone using the sequence from the wild-caught mosquitoes and characterized it via laboratory infections of mosquitoes and mosquito tissues.

Geographic Partitioning of Dengue Virus Transmission Risk in Florida.

Dengue viruses (DENVs) cause the greatest public health burden globally among the arthropod-borne viruses. DENV transmission risk has also expanded from tropical to subtropical regions due to the increasing range of its principal mosquito vector, Aedes aegypti. Focal outbreaks of dengue fever (dengue) in the state of Florida (FL) in the USA have increased since 2009. However, little is known about the competence of Ae. aegypti populations across different regions of FL to transmit DENVs. To understand the effects of DENV genotype and serotype variations on vector susceptibility and transmission potential in FL, we orally infected a colony of Ae. aegypti (Orlando/ORL) with low passage or laboratory DENV-1 through -4. Low passage DENVs were more infectious to and had higher transmission potential by ORL mosquitoes. We used these same DENVs to examine natural Ae. aegypti populations to determine whether spatial distributions correlated with differential vector competence. Vector competence across all DENV serotypes was greater for mosquitoes from areas with the highest dengue incidence in south FL compared to north FL. Vector competence for low passage DENVs was significantly higher, revealing that transmission risk is influenced by virus/vector combinations. These data support a targeted mosquito-plus-pathogen screening approach to more accurately estimate DENV transmission risk.

Isolation of SARS-CoV-2 from the air in a car driven by a COVID patient with mild illness.

OBJECTIVE: To determine if viable virus could be isolated from the air within a car driven by a patient infected with SARS-CoV-2, and to assess the size range of the infectious particles. METHODS: We used a Sioutas personal cascade impactor sampler (PCIS) to screen for SARS-CoV-2 in a car driven by a COVID-19 patient. The patient, who had only mild illness without fever or cough and was not wearing a mask, drove the car for 15 min with the air conditioning turned on and windows closed. The PCIS was clipped to the sun-visor above the front passenger seat and was retrieved from the car two hours after completion of the drive. RESULTS: SARS-CoV-2 was detectable at all PCIS stages by PCR and was cultured from the section of the sampler collecting particles in the 0.25-0.50 µm size range. CONCLUSIONS: Our data highlight the potential risk of SARS-CoV-2 transmission by minimally symptomatic persons in the closed space inside of a car and suggest that a substantial component of that risk is via aerosolized virus.

Earliest detection to date of SARS-CoV-2 in Florida: Identification together with influenza virus on the main entry door of a university building, February 2020.

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.

Emergence of porcine delta-coronavirus pathogenic infections among children in Haiti through independent zoonoses and convergent evolution.

Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, coronavirus emergence in our species has been associated with zoonotic transmissions from animal reservoirs 1,2 , underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae - Alphacoronavirus, Betacoronavirus, Deltacoronavirus, Gammacoronavirus , - human infections reported to date have been limited to alpha- and betacoronaviruses 3 . We identify, for the first time, porcine deltacoronavirus (PDCoV) strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the nsp15 and the spike glycoprotein genes by convergent evolution. In particular, structural analysis predicts that one of the changes in the Spike S1 subunit, which contains the receptor-binding domain, may affect protein's flexibility and binding to the host cell receptor. Our findings not only underscore the ability of deltacoronaviruses to adapt and potentially lead to human-to-human transmission, but also raise questions about the role of such transmissions in development of pre-existing immunity to other coronaviruses, such as SARS-CoV-2.

Orthobunyaviruses in the Caribbean: Melao and Oropouche virus infections in school children in Haiti in 2014.

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.