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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.

Keystone orthobunyavirus (KEYV), a member of the genus Orthobunyavirus, was first isolated in 1964 from mosquitoes in Keystone, Florida. Although data on human infections are limited, the virus has been linked to a fever/rash syndrome and, possibly, encephalitis, with early studies suggesting that 20% of persons in the Tampa, Florida, region had antibodies to KEYV. To assess the distribution and diversity of KEYV in other regions of Florida, we collected > 6,000 mosquitoes from 43 sampling sites in St. Johns County between June 2019 and April 2020. Mosquitoes were separated into pools by species and collection date and site. All pools with Aedes spp. (293 pools, 2,171 mosquitoes) were screened with a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay that identifies KEYV and other closely related virus species of what was previously designated as the California encephalitis serogroup. In 2020, screening for KEYV was expanded to include 211 pools of Culex mosquitoes from sites where KEYV-positive Aedes spp. had been identified. rRT-PCR–positive samples were inoculated into cell cultures, and five KEYV isolates from Aedes atlanticus pools were isolated and sequenced. Analyses of the KEYV large genome segment sequences revealed two distinct KEYV clades, whereas analyses of the medium and small genome segments uncovered past reassortment events. Our data documented the ongoing seasonal circulation of multiple KEYV clades within Ae. atlanticus mosquito populations along the east coast of Florida, highlighting the need for further studies of the impact of this virus on human health.

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., The presence of porcine deltacoronavirus has been detected in three children from Haiti that could have originated from zoonotic spillover.

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 minutes 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 to 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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain UF-8, with an in-frame 12-nucleotide deletion within open reading frame 3a (ORF3a), was isolated from a 78-year-old COVID-19 patient in March 2020.

We used a Sioutas personal cascade impactor sampler (PCIS) to screen for SARS-CoV-2 in a car driven by a COVID-19 patient. 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 to 0.50 □μm size range.

The progression of COVID-19 worldwide can be tracked by identifying mutations within the genomic sequence of SARS-CoV-2 that occur as a function of time. Such efforts currently rely on sequencing the genome of SARS-CoV-2 in patient specimens (direct sequencing) or of virus isolated from patient specimens in cell cultures. A pilot SARS-CoV-2 air sampling study conducted at a clinic within a university student health care center detected the virus vRNA, with an estimated concentration of 0.87 virus genomes L(−1) air. To determine whether the virus detected was viable (‘live’), attempts were made to isolate the virus in cell cultures. Virus-induced cytopathic effects (CPE) were observed within two days post-inoculation of Vero E6 cells with collection media from air samples; however, rtRT-PCR tests for SARS-CoV-2 vRNA from cell culture were negative. Instead, three other fast-growing human respiratory viruses were isolated and subsequently identified, illustrating the challenge in isolating SARS-CoV-2 when multiple viruses are present in a test sample. The complete SAR-CoV-2 genomic sequence was nevertheless determined by Sanger sequencing and most closely resembles SARS-CoV-2 genomes previously described in Georgia, USA. Results of this study illustrate the feasibility of tracking progression of the COVID-19 pandemic using environmental aerosol samples instead of human specimens. Collection of a positive sample from a distance more than 2 m away from the nearest patient traffic implies the virus was in an aerosol.

BackgroundQuestions persist about patterns of initial dissemination of SARS-CoV-2 in the United States in early 2020.MethodsIn 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. ResultsInfluenza H1N1pdm09 was isolated from the door handle on four of the 19 days sampled. Both SARS-CoV-2 and influenza virus were detected in the 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. ConclusionsOur 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.

Graphical abstract, Highlights • Viable (infectious) SARS-CoV-2 was present in aerosols within the hospital room of COVID-19 patients. • Airborne virus was detected in the absence of health-care aerosol-generating procedures. • The virus strain detected in the aerosols matched the virus strain isolated from a patient with acute COVID-19., Objectives Because detection of SARS-CoV-2 RNA in aerosols but failure to isolate viable (infectious) virus are commonly reported, there is substantial controversy whether SARS-CoV-2 can be transmitted through aerosols. This conundrum occurs because common air samplers can inactivate virions through their harsh collection processes. We sought to resolve the question whether viable SARS-CoV-2 can occur in aerosols using VIVAS air samplers that operate on a gentle water-vapor condensation principle. Methods Air samples collected in the hospital room of two COVID-19 patients, one ready for discharge, the other newly admitted, were subjected to RT-qPCR and virus culture. The genomes of the SARS-CoV-2 collected from the air and isolated in cell culture were sequenced. Results Viable SARS-CoV-2 was isolated from air samples collected 2 to 4.8 m away from the patients. The genome sequence of the SARS-CoV-2 strain isolated from the material collected by the air samplers was identical to that isolated from the newly admitted patient. Estimates of viable viral concentrations ranged from 6 to 74 TCID50 units/L of air. Conclusions Patients with respiratory manifestations of COVID-19 produce aerosols in the absence of aerosol-generating procedures that contain viable SARS-CoV-2, and these aerosols may serve as a source of transmission of the virus.

Background Beginning in December 2013, an epidemic of chikungunya virus (CHIKV) infection spread across the Caribbean and into virtually all countries in the Western hemisphere, with >2.4 million cases reported through the end of 2017. Methods We monitored a cohort of school children in rural Haiti from May 2014, through February 2015, for occurrence of acute undifferentiated febrile illness, with clinical and laboratory data available for 252 illness episodes. Results Our findings document passage of the major CHIKV epidemic between May and July 2014, with 82 laboratory-confirmed cases. Subsequent peaks of febrile illness were found to incorporate smaller outbreaks of dengue virus serotypes 1 and 4 and Zika virus, with identification of additional infections with Mayaro virus, enterovirus D68, and coronavirus NL63. CHIKV and dengue virus serotype 1 infections were more common in older children, with a complaint of arthralgia serving as a significant predictor for infection with CHIKV (odds ratio, 16.2; 95% confidence interval, 8.0–34.4; positive predictive value, 66%; negative predictive value, 80%). Conclusions Viral/arboviral infections were characterized by a pattern of recurrent outbreaks and case clusters, with the CHIKV epidemic representing just one of several arboviral agents moving through the population. Although clinical presentations of these agents are similar, arthralgias are highly suggestive of CHIKV infection., Data were obtained for 252 Haitian children with acute febrile illness from May 2014 through February 2015. Findings document passage of the major 2014 Chikungunya virus epidemic (82 laboratory-confirmed cases), as well as ongoing outbreaks/cases of Dengue, Zika, and Mayaro.

In January-April 2016, cattle and buffalo farm owners and veterinarians reported clinical signs suggestive of foot and mouth disease virus (FMDV) outbreaks among non-vaccinated cattle and buffalo herds in Egypt. The clinical disease observed was either mild (small oral lesions and speedy recovery) or severe (extensive oral lesions and/or mortalities), and the form of the disease (either mild or severe) segregated by farm. This study aimed to confirm the presence of FMDV and to characterize the circulating strains associated with the outbreaks. Vesicular epithelia were collected from 41 animals representing 15 affected cattle and buffalo farms in five governorates (Behira, Cairo, Daqahlia, Giza and Ismailia), and tested by real time (rt) RT-PCR. Consequently, 92% (38/41) of examined samples were positive. Furthermore, the VP1 coding region of 60% (23/38) of positive specimens were amplified by RT-PCR and sequenced. The phylogenetic analysis identified two distinct strains characterized as serotype O topotype EA-3 and serotype A (African topotype) of genotype IV in the severe and mild disease forms, respectively. The newly identified strains clustered in distinct clades in the phylogenetic trees, indicating the likelihood of new incursions into Egypt. Those strains were most closely related to previously described Sudanese strains.

Objectives To describe the epidemiology of malaria in pregnancy in Haiti. Methods Cross-sectional study among pregnant women in 6 departments of Haiti. After obtaining informed consent, whole blood samples and demographic surveys were collected to investigate malaria prevalence, anemia and socio-behavioral risk factors for infection, respectively. 311 pregnant women were screened for Plasmodium falciparum infection using a rapid diagnostic test (RDT), microscopy, and a novel, quantitative reverse-transcriptase polymerase chain reaction method (qRT-PCR). Results Overall, 1.2% (4/311) of pregnant women tested positive for malaria infection by both microscopy and RDT. However, using the qRT-PCR, 16.4% (51/311) of pregnant women were positive. The prevalence of malaria infection varied with geographic locations ranging between 0% to 46.4%. Additionally, 53% of pregnant women had some form of anemia; however no significant association was found between anemia and submicroscopic malaria infection. The socio-behavioral risk factors identified to be protective of malaria infection were marital status (P

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.

Though plans to eliminate malaria from the island of Hispaniola have recently received much attention, arbovirus surveillance continues to be largely neglected in Haiti. To support surveillance efforts and encourage vector-control strategies, a cross-sectional study of dengue virus (DENV) and West Nile virus (WNV) transmission was conducted using standard seroepidemiological methods. Blood samples (N = 673) were collected from 278 males and 395 females from three locations in the Ouest and Sud-Est Departments of Haiti. Serum was separated and tested for the presence of anti-DENV and anti-WNV immunoglobulin G (IgG) antibodies using an indirect enzyme-linked immunosorbent assay (ELISA). Anti-DENV IgG antibodies were detected in 72.1% (95% confidence interval [CI] = 68.7, 75.5) of the sample population; with no significant differences in seroprevalence by study location, participant gender, or age group (P > 0.1, in all tests). Anti-WNV IgG antibodies were detected in only 1% (95% CI = 0.3, 1.8) of the sample population, all which originated from participants located in Gressier. The high prevalence of anti-DENV IgG antibodies among all age groups, including those in the youngest age group (2–5 years of age), suggests hyperendemic transmission of DENV in the Ouest and Sud-Est Departments of Haiti. In contrast, the relative absence of anti-WNV IgG antibodies, even among older population members, further supports the notion that WNV transmission in this population is largely absent. These findings highlight the large burden of disease from DENV and the need for enhanced arbovirus surveillance and implementation of vector control strategies throughout Haiti.

Human coronavirus (HCoV) NL63 is recognized as a common cause of upper respiratory infections and influenza-like illness. In screening children with acute undifferentiated febrile illness in a school cohort in rural Haiti, we identified HCoV-NL63 in blood samples from four children. Cases clustered over an 11-day period; children did not have respiratory symptoms, but two had gastrointestinal complaints. On phylogenetic analysis, the Haitian HCoV-NL63 strains cluster together in a highly supported monophyletic clade linked most closely with recently reported strains from Malaysia; two respiratory HCoV-NL63 strains identified in north Florida in the same general period form a separate clade, albeit again with close linkages with the Malaysian strains. Our data highlight the variety of presentations that may be seen with HCoV-NL63, and underscore the apparent ease with which CoV strains move among countries, with our data consistent with recurrent introduction of strains into the Caribbean (Haiti and Florida) from Asia.

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.

We report the detection and isolation of enterovirus D68 from the blood of a 6-year-old child in rural Haiti, who presented with high fever and clinical signs suggestive of pneumonia. On phylogenetic analysis, this Haitian isolate was virtually identical to an enterovirus D68 strain circulating in New York during the same time period.

Human coronavirus strain 229E (HCoV-229E) and human alphaherpesvirus 1 were isolated from the plasma of a Haitian child in 2016 with suspected arbovirus diseases. To our knowledge, this is the first description of HCoV-229E in human plasma, which is the focus of this article.

An outbreak of dengue fever followed a chikungunya fever outbreak in Haiti in 2014. We detected Dengue virus 1 (DENV-1) in plasma samples collected between May 2014 and February 2015. A representative isolate was fully sequenced, and phylogenetic analyses indicate that it groups within the genotype V South American and Caribbean DENV-1 clades.

Background In Haiti where chloroquine (CQ) is widely used for malaria treatment, reports of resistance are scarce. However, recent identification of CQ resistance genotypes in one site is suggestive of an emerging problem. Additional studies are needed to evaluate genetic mutations associated with CQ resistance, especially in the Plasmodium falciparum multi-drug resistance- 1 gene (pfmdr 1) while expanding the already available information on P. falciparum CQ transporter gene (pfcrt) in Haiti. Methods Blood samples were collected on Whatman filter cards (FTA) from eight clinics spread across Haiti. Following the confirmation of P. falciparum in the samples, PCR protocols were used to amplify regions of pfmdr 1and pfcrt codons of interest, (86, 184, 1034, 1042, and 1246) and (72-76), respectively. Sequencing and site-specific restriction enzyme digestions were used to analyse these DNA fragments for the presence of single nucleotide polymorphisms (SNPs) known to confer resistance to anti-malarial drugs. Results P. falciparum infection was confirmed in160 samples by amplifying a segment of the P. falciparum 18S small subunit ribosomal RNA gene (pfssurrn a). The sequence of pfmdr1 in 54 of these samples was determined between codons 86,184 codons 1034, 1042 and 1246. No sequence differences from that of the NF54 clone 3D7 were found among the 54 samples except at codon 184, where a non-silent mutation was found in all samples predicted to alter the amino acid sequence replacing tyrosine with phenylalanine (Y184F). This altered sequence was also confirmed by restriction enzyme digestion. The sequence of pfmdr 1 at codons 86, 184, 1034 and 1042 encoded the NF SN haplotype. The sequence of pfcrt codons 72-76 from 79 samples was determined and found to encode CVMNK, consistent with a CQ sensitive genotype. Conclusion The presence of the Y184F mutation in pfmdr1 of P. falciparum parasites in Haiti may have implications for resistance to antimalarial drugs. The absence of mutation in pfcrt at codon 76 among 79 isolates tested suggests that sensitivity to CQ in Haiti remains common. Wide-spread screening of the pfmdr1 and pfcrt especially among patients experiencing treatment failure may be a useful tool in early detection of the emergence of antimalarial drug resistance in Haiti.

We analyzed highly pathogenic avian influenza A(H5N1) viruses isolated from humans infected in Egypt during 2007–2011. All analyzed viruses evolved from the lineage of subtype H5N1 viruses introduced into Egypt in 2006; we found minimal evidence of reassortment and no exotic introductions. The hemagglutinin genes of the viruses from 2011 formed a monophyletic group within clade 2.2.1 that also included human viruses from 2009 and 2010 and contemporary viruses from poultry; this finding is consistent with zoonotic transmission. Although molecular markers suggestive of decreased susceptibility to antiviral drugs were detected sporadically in the neuraminidase and matrix 2 proteins, functional neuraminidase inhibition assays did not identify resistant viruses. No other mutations suggesting a change in the threat to public health were detected in the viral proteomes. However, a comparison of representative subtype H5N1 viruses from 2011 with older subtype H5N1 viruses from Egypt revealed substantial antigenic drift.

Surveillance for influenza A viruses in wild birds has increased substantially as part of efforts to control the global movement of highly pathogenic avian influenza A (H5N1) virus. Studies conducted in Egypt from 2003 to 2007 to monitor birds for H5N1 identified multiple subtypes of low pathogenicity avian influenza A viruses isolated primarily from migratory waterfowl collected in the Nile Delta. Phylogenetic analysis of 28 viral genomes was performed to estimate their nearest ancestors and identify possible reassortants. Migratory flyway patterns were included in the analysis to assess gene flow between overlapping flyways. Overall, the viruses were most closely related to Eurasian, African and/or Central Asian lineage low pathogenicity viruses and belonged to 15 different subtypes. A subset of the internal genes seemed to originate from specific flyways (Black Sea-Mediterranean, East African-West Asian). The remaining genes were derived from a mixture of viruses broadly distributed across as many as 4 different flyways suggesting the importance of the Nile Delta for virus dispersal. Molecular clock date estimates suggested that the time to the nearest common ancestor of all viruses analyzed ranged from 5 to 10 years, indicating frequent genetic exchange with viruses sampled elsewhere. The intersection of multiple migratory bird flyways and the resulting diversity of influenza virus gene lineages in the Nile Delta create conditions favoring reassortment, as evident from the gene constellations identified by this study. In conclusion, we present for the first time a comprehensive phylogenetic analysis of full genome sequences from low pathogenic avian influenza viruses circulating in Egypt, underscoring the significance of the region for viral reassortment and the potential emergence of novel avian influenza A viruses, as well as representing a highly diverse influenza A virus gene pool that merits continued monitoring.

Migratory (particularly aquatic) birds are the major natural reservoirs for type A influenza viruses. However, their role in transmitting highly pathogenic avian influenza (HPAI) viruses is unclear. Egypt is a "funnel" zone of wild bird migration pathways from Central Asia and Europe to Eastern and Central Africa ending in South Africa. We sought to detect and isolate avian influenza viruses in migratory birds in Egypt. During September 2003-February 2009, the US Naval Medical Research Unit Number 3, Cairo, Egypt, in collaboration with the Egyptian Ministry of Environment, obtained cloacal swabs from 7,894 migratory birds captured or shot by hunters in different geographic areas in Egypt. Samples were processed by real-time reverse transcriptase PCR for detection of the influenza A matrix gene. Positive samples were processed for virus isolation in specific-pathogen-free embryonated eggs and isolates were subtyped by PCR and partial sequencing. Ninety-five species of birds were collected. Predominant species were Green-Winged Teal (Anas carolinensis; 32.0%, n=2,528), Northern Shoveler (Anas clypeata; 21.4%, n=1,686), and Northern Pintail (Anas acuta; 11.1%, n=877). Of the 7,894 samples, 745 (9.4%) were positive for the influenza A matrix gene (mainly from the above predominant species). Thirteen of the 745 (1.7%) were H5-positive by PCR (11 were low-pathogenic avian influenza and two were HPAI H5N1). The prevalences of influenza A was among regions were 10-15%, except in Middle Egypt (4%). Thirty-nine influenza isolates were obtained from PCR-positive samples. Seventeen subtypes of avian influenza viruses (including H5N1 and H7N7) were classified from 39 isolates using PCR and partial sequencing. Only one HPAI H5N1 was isolated in February 2006, from a wild resident Great Egret (Ardea alba). No major die-offs or sick migratory birds were detected during the study. We identified avian influenza virus subtypes not previously reported in Egypt. The HPAI H5N1 isolated or detected indicates that migratory birds may play a role in the dispersal of HPAI virus, but a detailed mechanism of this role needs to be elucidated.