DNA Barcoding Analysis of Trinidad Haemagogus Mosquitoes Reveals Evidence for Putative New Species.

Background: Haemagogus janthinomys is a primary sylvan vector of yellow fever virus and the emerging Mayaro virus. However, despite its medical importance, there is a dearth of data on the molecular taxonomy of this mosquito species. Methods: In this study, DNA barcoding analysis was performed on 64 adult female mosquitoes from Trinidad morphologically identified as Hg. janthinomys. The mitochondrial cytochrome c oxidase I (COI) gene and ribosomal DNA internal transcribed spacer 2 (ITS2) region of the mosquitoes were PCR amplified and sequenced, and molecular phylogenies inferred. Results: The BLASTN analysis showed that only 20% (n = 13/66) of COI sequences had high similarity (>99% identity) to Hg. janthinomys and the remaining sequences had low similarity (<90% identity) to reference GenBank sequences. Phylogenetic analysis of COI sequences revealed the presence of four strongly supported groups, with one distinct clade that did not align with any reference sequences. Corresponding ITS2 sequences for samples in this distinct COI group clustered into three clades. Conclusions: These molecular findings suggest the existence of a putative new Haemagogus mosquito species and underscore the need for further, more in-depth investigations into the taxonomy and classification of the Haemagogus genus.

Chikungunya: a decade of burden in the Americas.

In the Americas, one decade following its emergence in 2013, chikungunya virus (CHIKV) continues to spread and cause epidemics across the region. To date, 3.7 million suspected and laboratory-confirmed chikungunya cases have been reported in 50 countries or territories in the Americas. Here, we outline the current status and epidemiological aspects of chikungunya in the Americas and discuss prospects for future research and public health strategies to combat CHIKV in the region.

Toward a global virus genomic surveillance network.

The COVID-19 pandemic galvanized the field of virus genomic surveillance, demonstrating its utility for public health. Now, we must harness the momentum that led to increased infrastructure, training, and political will to build a sustainable global genomic surveillance network for other epidemic and endemic viruses. We suggest a generalizable modular sequencing framework wherein users can easily switch between virus targets to maximize cost-effectiveness and maintain readiness for new threats. We also highlight challenges associated with genomic surveillance and when global inequalities persist. We propose solutions to mitigate some of these issues, including training and multilateral partnerships. Exploring alternatives to clinical sequencing can also reduce the cost of surveillance programs. Finally, we discuss how establishing genomic surveillance would aid control programs and potentially provide a warning system for outbreaks, using a global respiratory virus (RSV), an arbovirus (dengue virus), and a regional zoonotic virus (Lassa virus) as examples.

Phylogenetic characterization of Orthobunyaviruses isolated from Trinidad shows evidence of natural reassortment.

The genus Orthobunyavirus is a diverse group of viruses in the family Peribunyaviridae, recently classified into 20 serogroups, and 103 virus species. Although most viruses within these serogroups are phylogenetically distinct, the absence of complete genome sequences has left several viruses incompletely characterized. Here we report the complete genome sequences for 11 orthobunyaviruses isolated from Trinidad, French Guiana, Guatemala, and Panama that were serologically classified into six serogroups and 10 species. Phylogenetic analyses of these 11 newly derived sequences indicate that viruses belonging to the Patois, Capim, Guama, and Group C serocomplexes all have a close genetic origin. We show that three of the 11 orthobunyaviruses characterized (belonging to the Group C and Bunyamwera serogroups) have evidence of histories of natural reassortment through the M genome segment. Our data also suggests that two distinct lineages of Group C viruses concurrently circulate in Trinidad and are transmitted by the same mosquito vectors. This study also highlights the importance of complementing serological identification with nucleotide sequencing when characterizing orthobunyaviruses.

Saliva-based methods for SARS-CoV-2 testing in low- and middle-income countries.

As the coronavirus disease 2019 (COVID-19) continues to disproportionately affect low- and middle-income countries, the need for simple, accessible and frequent diagnostic testing grows. In lower-resource settings, case detection is often limited by a lack of available testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address global inequities in testing, alternative sample types could be used to increase access to testing by reducing the associated costs. Saliva is a sensitive, minimally invasive and inexpensive diagnostic sample for SARS-CoV-2 detection that is appropriate for asymptomatic surveillance, symptomatic testing and at-home collection. Saliva testing can lessen two major challenges faced by lower- and middle-income countries: constrained resources and overburdened health workers. Saliva sampling enables convenient self-collection and requires fewer resources than swab-based methods. However, saliva testing for SARS-CoV-2 diagnostics has not been implemented on a large scale in low- and middle-income countries. While numerous studies based in these settings have demonstrated the usefulness of saliva sampling, there has been insufficient attention on optimizing its implementation in practice. We argue that implementation science research is needed to bridge this gap between evidence and practice. Low- and middle-income countries face many barriers as they continue their efforts to provide mass COVID-19 testing in the face of substantial inequities in global access to vaccines. Laboratories should look to replicate successful approaches for sensitive detection of SARS-CoV-2 in saliva, while governments should act to facilitate mass testing by lifting restrictions that limit implementation of saliva-based methods.

La maladie à coronavirus 2019 (COVID-19) continue à affecter les pays à revenu faible et intermédiaire de manière disproportionnée, accentuant le besoin en tests diagnostiques simples, accessibles et fréquents. Dans les endroits disposant de ressources limitées, la détection des cas se heurte souvent au manque de tests disponibles pour le syndrome respiratoire aigu sévère (SARS-CoV-2). Afin de lutter contre les inégalités mondiales en la matière, d'autres types d'échantillons pourraient être exploités, dans le but d'améliorer l'accès au dépistage tout en diminuant les frais qu'il engendre. Les échantillons de salive offrent une méthode de diagnostic fiable, peu invasive et peu coûteuse pour détecter le SARS-CoV-2. Cette méthode est compatible avec le suivi des personnes asymptomatiques, le dépistage des personnes symptomatiques et la collecte d'échantillons à domicile. Les tests salivaires permettent d'atténuer deux problèmes majeurs rencontrés par les pays à revenu faible et intermédiaire: une pénurie de ressources et des soignants surmenés. En outre, les patients peuvent effectuer le prélèvement eux-mêmes et cette méthode nécessite moins de moyens que celle reposant sur l'écouvillonnage. Pourtant, les tests salivaires de détection du SARS-CoV-2 n'ont pas été déployés à grande échelle dans les pays à revenu faible et intermédiaire. Malgré les nombreuses études démontrant l'utilité des tests salivaires dans ces régions, les perspectives d'optimisation de leur mise en œuvre n'ont suscité que peu d'attention. Dans le présent document, nous affirmons que des recherches scientifiques sur leur exécution sont requises pour combler ce fossé entre les faits et la pratique. Les pays à revenu faible et intermédiaire sont confrontés à une multitude d'obstacles dans leurs efforts de dépistage massif de la COVID-19. Et ce, en dépit des profondes inégalités qu'ils subissent dans le monde en matière d'accès aux vaccins. Les laboratoires devraient tenter de reproduire les approches les plus efficaces pour détecter le SARS-CoV-2 dans la salive, tandis que les gouvernements devraient prendre des mesures favorisant un dépistage de masse en levant les restrictions qui entravent le déploiement des tests salivaires.

A medida que la enfermedad por coronavirus de 2019 (COVID-19) sigue afectando de manera desproporcionada a los países de ingresos bajos y medios, crece la necesidad de realizar pruebas de diagnóstico sencillas, accesibles y frecuentes. En entornos de bajos recursos, la detección de casos suele estar limitada por la falta de pruebas disponibles para diagnosticar el coronavirus del síndrome respiratorio agudo grave de tipo 2 (SARS-CoV-2). Para abordar las desigualdades globales en las pruebas, se podrían utilizar tipos de muestra alternativos para aumentar el acceso a las pruebas reduciendo los costes asociados. La saliva es una muestra de diagnóstico sensible, poco invasiva y económica para la detección del SARS-CoV-2 que es apropiada para la vigilancia asintomática, las pruebas sintomáticas y la obtención en el hogar. Las pruebas de saliva pueden reducir dos de los principales problemas a los que se enfrentan los países de ingresos bajos y medios: la escasez de recursos y la sobrecarga de trabajo del personal sanitario. La toma de muestras de saliva permite realizar fácilmente la obtención por cuenta propia y requiere menos recursos que los métodos con hisopos. Sin embargo, las pruebas de saliva para el diagnóstico del SARS-CoV-2 no se han aplicado a gran escala en los países de ingresos bajos y medios. Aunque varios estudios realizados en estos entornos han demostrado la utilidad del muestreo de saliva, no se ha prestado suficiente atención a la optimización de su aplicación en la práctica. En este sentido, se considera que la investigación científica sobre la implementación es necesaria para subsanar esta deficiencia entre la evidencia y la práctica. Los países de ingresos bajos y medios se enfrentan a muchas dificultades en sus esfuerzos por realizar pruebas masivas en relación con la COVID-19, a pesar de las grandes desigualdades en el acceso global a las vacunas. Los laboratorios deberían intentar reproducir los enfoques que han tenido éxito para la detección sensible de la infección por el SARS-CoV-2 en la saliva, mientras que los gobiernos deberían actuar para facilitar las pruebas masivas eliminando las restricciones que limitan la aplicación de los métodos de diagnóstico salival.

Global disparities in SARS-CoV-2 genomic surveillance.

Genomic sequencing is essential to track the evolution and spread of SARS-CoV-2, optimize molecular tests, treatments, vaccines, and guide public health responses. To investigate the global SARS-CoV-2 genomic surveillance, we used sequences shared via GISAID to estimate the impact of sequencing intensity and turnaround times on variant detection in 189 countries. In the first two years of the pandemic, 78% of high-income countries sequenced >0.5% of their COVID-19 cases, while 42% of low- and middle-income countries reached that mark. Around 25% of the genomes from high income countries were submitted within 21 days, a pattern observed in 5% of the genomes from low- and middle-income countries. We found that sequencing around 0.5% of the cases, with a turnaround time <21 days, could provide a benchmark for SARS-CoV-2 genomic surveillance. Socioeconomic inequalities undermine the global pandemic preparedness, and efforts must be made to support low- and middle-income countries improve their local sequencing capacity.

A high-throughput screening assay to identify inhibitory antibodies targeting alphavirus release.

BACKGROUND: Several studies have demonstrated neutralizing antibodies to be highly effective against alphavirus infection in animal models, both prophylactically and remedially. In most studies, neutralizing antibodies have been evaluated for their ability to block viral entry in vitro but recent evidence suggests that antibody inhibition through other mechanisms, including viral budding/release, significantly contributes to viral control in vivo for a number of alphaviruses. RESULTS: We describe a BSL-2, cell-based, high-throughput screening system that specifically screens for inhibitors of alphavirus egress using chikungunya virus (CHIKV) and Mayaro virus (MAYV) novel replication competent nano-luciferase (nLuc) reporter viruses. Screening of both polyclonal sera and memory B-cell clones from CHIKV immune individuals using the optimized assay detected several antibodies that display potent anti-budding activity. CONCLUSIONS: We describe an "anti-budding assay" to specifically screen for inhibitors of viral egress using novel CHIKV and MAYV nLuc reporter viruses. This BSL-2 safe, high-throughput system can be utilized to explore neutralizing "anti-budding" antibodies to yield potent candidates for CHIKV and MAYV therapeutics and prophylaxis.

Comparison of surveillance trapping methods to monitor Culicoides biting midge activity in Trinidad, West Indies.

Culicoides biting midges (Diptera: Ceratopogonidae) are biting nuisances and arbovirus vectors of both public health and veterinary significance in Trinidad. We compared sampling methods to define the behaviour and bionomics of adult Culicoides populations at a commercial dairy goat farm. Three static trap designs were compared: (a) Centre for Disease Control (CDC) downdraft UV trap; (b) CDC trap with an incandescent bulb and (c) CDC trap with semiochemical lure consisting of R-(-)-1-octen-3-ol and CO2 (no bulb). Sweep netting was used to define diel periodicity. A total of 30,701 biting midges were collected using static traps, dominated by female Culicoides furens (>70% of trap collections across all three designs). There was no significant difference in the Margalef's index between the three traps; however, trap designs A and C collected a significantly greater number of individuals than trap B, and trap C gained highest species richness. The greatest species richness and abundance of Culicoides collected by sweep net was observed between 6:00 and 6:15 pm and notable differences in the crepuscular activity pattern of several species were identified. Comparative data on Culicoides species richness, abundance, sex and reproductive status is discussed and can be used to improve surveillance strategies, research designs and risk management.

Genomics-informed outbreak investigations of SARS-CoV-2 using civet.

The scale of data produced during the SARS-CoV-2 pandemic has been unprecedented, with more than 13 million sequences shared publicly at the time of writing. This wealth of sequence data provides important context for interpreting local outbreaks. However, placing sequences of interest into national and international context is difficult given the size of the global dataset. Often outbreak investigations and genomic surveillance efforts require running similar analyses again and again on the latest dataset and producing reports. We developed civet (cluster investigation and virus epidemiology tool) to aid these routine analyses and facilitate virus outbreak investigation and surveillance. Civet can place sequences of interest in the local context of background diversity, resolving the query into different 'catchments' and presenting the phylogenetic results alongside metadata in an interactive, distributable report. Civet can be used on a fine scale for clinical outbreak investigation, for local surveillance and cluster discovery, and to routinely summarise the virus diversity circulating on a national level. Civet reports have helped researchers and public health bodies feedback genomic information in the appropriate context within a timeframe that is useful for public health.

Global disparities in SARS-CoV-2 genomic surveillance.

Genomic sequencing provides critical information to track the evolution and spread of SARS-CoV-2, optimize molecular tests, treatments and vaccines, and guide public health responses. To investigate the spatiotemporal heterogeneity in the global SARS-CoV-2 genomic surveillance, we estimated the impact of sequencing intensity and turnaround times (TAT) on variant detection in 167 countries. Most countries submit genomes >21 days after sample collection, and 77% of low and middle income countries sequenced <0.5% of their cases. We found that sequencing at least 0.5% of the cases, with a TAT <21 days, could be a benchmark for SARS-CoV-2 genomic surveillance efforts. Socioeconomic inequalities substantially impact our ability to quickly detect SARS-CoV-2 variants, and undermine the global pandemic preparedness.

Characterization of the virome associated with Haemagogus mosquitoes in Trinidad, West Indies.

Currently, there are increasing concerns about the possibility of a new epidemic due to emerging reports of Mayaro virus (MAYV) fever outbreaks in areas of South and Central America. Haemagogus mosquitoes, the primary sylvan vectors of MAYV are poorly characterized and a better understanding of the mosquito's viral transmission dynamics and interactions with MAYV and other microorganisms would be important in devising effective control strategies. In this study, a metatranscriptomic based approach was utilized to determine the prevalence of RNA viruses in field-caught mosquitoes morphologically identified as Haemagogus janthinomys from twelve (12) forest locations in Trinidad, West Indies. Known insect specific viruses including the Phasi Charoen-like and Humaiata-Tubiacanga virus dominated the virome of the mosquitoes throughout sampling locations while other viruses such as the avian leukosis virus, MAYV and several unclassified viruses had a narrower distribution. Additionally, assembled contigs from the Ecclesville location suggests the presence of a unique uncharacterized picorna-like virus. Mapping of RNA sequencing reads to reference mitochondrial sequences of potential feeding host animals showed hits against avian and rodent sequences, which putatively adds to the growing body of evidence of a potentially wide feeding host-range for the Haemagogus mosquito vector.

Isolation of a novel insect-specific flavivirus with immunomodulatory effects in vertebrate systems.

We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.

Novel quaranjavirus and other viral sequences identified from ticks parasitizing hunted wildlife in Trinidad and Tobago.

Hunters are at a higher risk for exposure to zoonotic pathogens due to their close interactions with wildlife and arthropod vectors. In this study, high throughput sequencing was used to explore the viromes of two tick species, Amblyomma dissimile and Haemaphysalis juxtakochi, removed from hunted wildlife in Trinidad and Tobago. We identified sequences from 3 new viral species, from the viral families Orthomyxoviridae, Chuviridae and Tetraviridae in A. dissimile.

Generation of recombinant hyperimmune globulins from diverse B-cell repertoires.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

Characterization of novel, pathogenic field strains of infectious bronchitis virus (IBV) in poultry in Trinidad and Tobago.

Avian coronaviruses, including infectious bronchitis virus (IBV) and turkey coronavirus (TCoV), are economically important viruses affecting poultry worldwide. IBV is responsible for causing severe losses to the commercial poultry sector globally. The objectives of this study were to identify the viruses that were causing outbreaks of severe respiratory disease in chickens in Trinidad and Tobago (T&T) and to characterize the strains. Swab samples were collected from birds showing severe respiratory signs in five farms on the island of Trinidad. Samples were tested for the presence of IBV, as well as avian influenza virus (AIV), Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) by real-time reverse transcription polymerase chain reaction (qRT-PCR). All samples from the five farms tested negative for AIV, NDV and aMPV; however, samples from clinically affected birds in all five of the farms tested positive for IBV. Genetic data revealed the presence of TCoV in chickens on two of the farms. Interestingly, these two farms had never reared turkeys. Phylogenetic analysis showed that IBV S1 sequences formed two distinct clusters. Two sequences grouped with vaccine strains within the GI-1 lineage, whereas three sequences grouped together, but separately from other defined lineages, forming a likely new lineage of IBV. Pairwise comparison revealed that the three unique variant strains within the distinct lineage of IBV were significantly different in their S1 nucleotide coding regions from viruses in the closest lineage (16% difference) and locally used vaccine strains (>20% difference). Results also suggested that one of the samples was a recombinant virus, generated from a recombination event between a Trinidad virus of the GI-1 lineage and a Trinidad virus of the newly defined lineage. Many amino acid differences were also observed between the S1 coding regions of the circulating field and vaccine strains, indicating that the IBV vaccines may not be protective. Vaccine-challenge studies are however needed to prove this.

Serological Evidence for Henipa-like and Filo-like Viruses in Trinidad Bats.

Bat-borne zoonotic pathogens belonging to the family Paramxyoviridae, including Nipah and Hendra viruses, and the family Filoviridae, including Ebola and Marburg viruses, can cause severe disease and high mortality rates on spillover into human populations. Surveillance efforts for henipaviruses and filoviruses have been largely restricted to the Old World; however, recent studies suggest a potentially broader distribution for henipaviruses and filoviruses than previously recognized. In the current study, we screened for henipaviruses and filoviruses in New World bats collected across 4 locations in Trinidad near the coast of Venezuela. Bat tissue samples were screened using previously established reverse-transcription polymerase chain reaction assays. Serum were screened using a multiplex immunoassay to detect antibodies reactive with the envelope glycoprotein of viruses in the genus Henipavirus and the family Filoviridae. Serum samples were also screened by means of enzyme-linked immunosorbent assay for antibodies reactive with Nipah G and F glycoproteins. Of 84 serum samples, 28 were reactive with ≥1 henipavirus glycoprotein by ≥1 serological method, and 6 serum samples were reactive against ≥1 filovirus glycoproteins. These data provide evidence of potential circulation of viruses related to the henipaviruses and filoviruses in New World bats.

The Serological Prevalence of Rabies Virus-Neutralizing Antibodies in the Bat Population on the Caribbean Island of Trinidad.

: Rabies virus (RABV) is the only lyssavirus known to be present within the Caribbean. The island of Trinidad, is richly diverse in chiropteran fauna and endemic for bat-transmitted rabies with low RABV isolation rates observed in this population. We aimed to determine the seroprevalence of rabies virus neutralizing antibodies (RVNA) in light of spatio-temporal and bat demographic factors to infer the extent of natural exposure to RABV in the Trinidadian bat population. RVNA titers were determined by the RABV micro-neutralization test on 383 bat samples representing 21 species, comprising 30.9% of local bat diversity, from 31 locations across the island over 5 years. RVNA was positively detected in 33 samples (8.6%) representing 6 bat species (mainly frugivorous) with titers ranging from 0.1 to 19 IU/mL (mean 1.66 IU/mL). The analyses based on a multivariable binomial generalised linear mixed-effects model showed that bat age and year of capture were significant predictors of seropositivity. Thus, juvenile bats were more likely to be seropositive when compared to adults (estimate 1.13; p = 0.04) which may suggest early exposure to the RABV with possible implications for viral amplification in this population. Temporal variation in rabies seropositivity, 2012-2014 versus 2015-2017 (estimate 1.07; p = 0.03) may have been related to the prevailing rabies epizootic situation. Regarding other factors investigated, RVNA was found in bats from both rural and non-rural areas, as well as in both hematophagous and non-hematophagous bat species. The most common seropositive species, Artibeusjamaicensisplanirostris is ubiquitous throughout the island which may potentially facilitate human exposure. The findings of this study should be factored into public health assessments on the potential for rabies transmission by non-hematophagous bats in Trinidad.

Determinants of dengue virus dispersal in the Americas.

Dengue viruses (DENVs) are classified into four serotypes, each of which contains multiple genotypes. DENV genotypes introduced into the Americas over the past five decades have exhibited different rates and patterns of spatial dispersal. In order to understand factors underlying these patterns, we utilized a statistical framework that allows for the integration of ecological, socioeconomic, and air transport mobility data as predictors of viral diffusion while inferring the phylogeographic history. Predictors describing spatial diffusion based on several covariates were compared using a generalized linear model approach, where the support for each scenario and its contribution is estimated simultaneously from the data set. Although different predictors were identified for different serotypes, our analysis suggests that overall diffusion of DENV-1, -2, and -3 in the Americas was associated with airline traffic. The other significant predictors included human population size, the geographical distance between countries and between urban centers and the density of people living in urban environments.