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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.

Objectives: Because the detection of SARS-CoV-2 RNA in aerosols but failure to isolate viable (infectious) virus are commonly reported, there is substantial controversy whether severe acute respiratory syndrome coronavirus 2 (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 coronavirus disease-2019 (COVID-19) patients, one ready for discharge and 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.

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.

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.

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.

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.

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.

Malaria is an important health burden in Zambia with proper diagnosis remaining as one of the biggest challenges. The need for reliable diagnostics is being addressed through the introduction of rapid diagnostic tests (RDTs). However, without sufficient laboratory amenities in many parts of the country, diagnosis often still relies on non-specific, clinical symptoms. In this study, geographical information systems were used to both visualize and analyze the spatial distribution and the risk factors related to the diagnosis of malaria. The monthly reported, district-level number of malaria cases from January 2009 to December 2014 were collected from the National Malaria Control Center (NMCC). Spatial statistics were used to reveal cluster tendencies that were subsequently linked to possible risk factors, using a non-spatial regression model. Significant, spatio-temporal clusters of malaria were spotted while the introduction of RDTs made the number of clinically diagnosed malaria cases decrease by 33% from 2009 to 2014. The limited access to road network(s) was found to be associated with higher levels of malaria, which can be traced by the expansion of health promotion interventions by the NMCC, indicating enhanced diagnostic capability. The capacity of health facilities has been strengthened with the increased availability of proper diagnostic tools and through retraining of community health workers. To further enhance spatial decision support systems, a multifaceted approach is required to ensure mobilization and availability of human, infrastructural and technological resources. Surveillance based on standardized geospatial or other analytical methods should be used by program managers to design, target, monitor and assess the spatio-temporal dynamics of malaria diagnostic resources country-wide.

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.