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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. National Institute of Allergy and Infectious Diseases of the National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Allergy & Infectious Diseases (NIAID) [K22AI125474, R01AI153433, R01AI121452, R24AI120942] Published version This work was supported by grants from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers K22AI125474 and R01AI153433 to AJA, and R01AI121452 and R24AI120942 to SCW. We thank Steven Widen, Thomas Wood and Jill Thompson for assistance with sequencing.

Treatment of cancers in the lung remains a critical challenge in the clinic for which gene therapy could offer valuable options. We describe an effective approach through systemic injection of engineered polymer/DNA nanoparticles that mediate tumor-specific expression of a therapeutic gene, under the control of the cancer-selective progression elevated gene 3 (PEG-3) promoter, to treat tumors in the lungs of diseased mice. A clinically tested, untargeted, polyethylenimine carrier was selected to aid rapid transition to clinical studies, and a CpG-free plasmid backbone and coding sequences were used to reduce inflammation. Intravenous administration of nanoparticles expressing murine single-chain interleukin 12, under the control of PEG-3 promoter, significantly improved the survival of mice in both an orthotopic and a metastatic model of lung cancer with no marked symptoms of systemic toxicity. These outcomes achieved using clinically relevant nanoparticle components raises the promise of translation to human therapy.

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 thousands-diverse mixtures 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 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 three 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 (ATG), we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

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 (TT) 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.

Ticks are vectors of a wide variety of pathogens that are implicated in mild to severe disease in humans and other animals. Nonetheless, the full range of tick-borne pathogens is unknown. Viruses, in particular, have been neglected in discovery efforts targeting tick-borne agents. High throughput sequencing was used to characterize the virome of 638 ticks, including Rhipicephalus microplus (n = 320), Rhipicephalus sanguineus (n = 300), and Amblyomma ovale (n = 18) collected throughout Trinidad and Tobago in 2017 and 2018. Sequences representing nine viruses were identified, including five novel species within Tymovirales, Bunyavirales, Chuviridae, Rhabdoviridae, and Flaviviridae. Thereafter the frequency of detection of viral sequences in individual tick species was investigated.

Highlights • Epizootic haemorrhagic disease virus serotype 6 (EHDV-6) is circulating in Trinidad. • EHDV is infecting cattle at a slower rate than BTV. • EHDV appears to have a faster viral evolution rate than BTV. • The EHDV-6 Trinidad strain (VP-2) falls within the eastern topotype clade that is likely to have originated from Australia., Epizootic hemorrhagic disease virus (EHDV) is an economically important virus that can cause severe clinical disease in deer and to a lesser extent cattle. This study set out to determine and characterize which EHDV serotypes were circulating in Trinidad. Serum and whole blood samples were collected monthly for six months from a cohort of cattle imported to Trinidad from the USA. Results revealed that all the cattle seroconverted to EHDV within six months of their arrival, with EHDV RNA being detected in the samples just prior to antibodies, as expected. Serotyping assays revealed that a single serotype (EHDV-6) was circulating in the cattle. Sequencing of the surface viral protein (VP2) of EHDV-6, followed by phylogenetic analysis, revealed that the Trinidad EHDV-6 strain was closely related to EHDV-6 viruses found in Guadeloupe (2010), Martinique (2010) and USA (2006), with 96–97.2% nucleotide identity. The Trinidad EHDV-6 VP-2 shared 97.2% identity with the Australian EHDV-6 prototype strain, classifying it within the eastern topotype clade. Bayesian coalescent analysis support Australia as the most probable source for the EHDV-6 VP2 sequences in the Americas and Caribbean region and suggests that the they diverged from the Australian prototype strain around 1966 (95% HPD 1941–1979).

Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501YV2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected., Wellcome Open Research, 6

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 Chaoren-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 partial or complete mitochondrial genomes showed hits against avian and rodent genomes, which putatively add to the growing body of evidence of a potentially wide feeding host-range for the Haemagogus mosquito vector.

Plasma-derived polyclonal antibodies are polyvalent drugs used for many important clinical indications that require modulation of multiple drug targets simultaneously, including emerging infectious disease and transplantation. However, plasma-derived drugs suffer many problems, including low potency, impurities, constraints on supply, and batch-to-batch variation. In this study, we demonstrated proofs-of-concept for a technology that uses microfluidics and molecular genomics to capture diverse mammalian antibody repertoires as multivalent recombinant drugs. These “recombinant hyperimmune” drugs comprised thousands to tens of thousands of antibodies and were derived from convalescent human donors, or vaccinated human donors or immunized mice. Here we used our technology to build a highly potent recombinant hyperimmune for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2) in less than three months. We also validated a recombinant hyperimmune for Zika virus disease that abrogates antibody-dependent enhancement (ADE) through Fc engineering. For patients with primary immune deficiency (PID), we built high potency polyvalent recombinant hyperimmunes against pathogens that commonly cause serious lung infections. Finally, to address the limitations of rabbit-derived anti-thymocyte globulin (ATG), we generated a recombinant human version and demonstrated in vivo function against graft-versus-host disease (GVHD). Recombinant hyperimmunes are a novel class of drugs that could be used to target a wide variety of other clinical applications, including cancer and autoimmunity.

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. ispartof: VIRUS EVOLUTION vol:6 issue:2 ispartof: location:England status: published

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.

Viruses affecting poultry cause significant levels of disease leading to severe economic losses among poultry farmers worldwide. The Americas region continues to be vulnerable to the spread of poultry viruses across the continents and Caribbean island chains. In Trinidad and Tobago (T&T) there is limited information on the viruses circulating in poultry. Many flock are vulnerable to infection and there are occasional outbreaks of disease resulting in high levels of morbidity and mortality. This study aims to identify important viruses of poultry circulating in T&T through a broad-based surveillance approach. Serum samples from 29 layer farms in Trinidad and 14 layer farms in Tobago were collected from the eldest laying hens. Samples were tested from unvaccinated birds for antibodies by enzyme-linked immunosorbent assay (ELISA) against Avian influenza virus (AIV), Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Infectious laryngotracheitis virus (ILTV), Avian pneumovirus (APV), Infectious bursal disease virus (IBDV), Fowl adenovirus Gp1 (FADV) and Egg drop syndrome virus (EDSV). In Trinidad, the estimated true seroprevalence levels of antibodies were 0% (CI 95%: 0-0%) for AIV, 100% (CI 95%: 97-100%) for IBV, 79.8% (CI 95%: 70.6-86.9%) for NDV, 1% (CI 95%: 0-2.6%) for ILTV, 67.55% (CI 95%: 62.3-72.4%) for APV, 94.93% (CI 95%: 88.0-98.6%) for IBDV, 100% (CI 95%: 99.7-100%) for FADV and 67.8% (CI 95%: 62.4-72.8%) for EDSV. In Tobago, seroprevalence levels were 0% (CI 95%: 0-0%) for AIV, 100% (CI 95%: 95.6-100%) for IBV, 80.5% (CI 95%: 70.1-88.5%) for NDV, 29.9% (CI 95%: 20.8-40.6%) for ILTV, 100% (CI 95%: 97.7-100%) for APV, 97.1% (CI95%: 89.9-100%) for IBDV, 100% (CI 95%: 97.5-100%) for FADV and 100% (CI 95%: 99-100%) for EDSV. The results reveal strong evidence for the circulation of IBV, NDV, APV, IBDV, FADV and EDSV in layer poultry on both islands, as well as ILTV in 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.

The objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (SwIV), as well as the seroprevalence of porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) in pigs in Trinidad and Tobago (T&T). Blood samples (309) were randomly collected from pigs at farms throughout T&T. Serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial ELISA kits, and the circulating strains of SwIV were identified by the hemagglutination inhibition test (HIT). Antibodies against SwIV were detected in 114 out of the 309 samples (37%). Out of a total of 26 farms, 14 tested positive for SwIV antibodies. HI testing revealed high titers against the A/sw/Minnesota/593/99 H3N2 strain and the pH1N1 2009 pandemic strain. Antibodies against PPV were detected in 87 out of the 309 samples (28%), with 11 out of 26 farms testing positive for PPV antibodies. Antibodies against PCV-2 were detected in 205 out of the 309 samples tested (66%), with 25 out of the 26 farms testing positive for PCV-2 antibodies. No antibodies were detected in any of the tested pigs to PRRSV, TGEV, PRCV, or CSFV.

Purpose: This paper describes the rationale, study design and procedures of the National Eye Survey of Trinidad and Tobago (NESTT). The main objective of this survey is to obtain prevalence estimates of vision impairment and blindness for planning and policy development.\ud \ud Methods: A population-based, cross-sectional survey was undertaken using random multistage cluster sampling, with probability-proportionate-to-size methods. Eligible participants aged 5 years and older were sampled from the non-institutional population in each of 120 cluster segments. Presenting distance and near visual acuity were screened in their communities. People aged 40 years and older, and selected younger people, were invited for comprehensive clinic assessment. The interview included information on potential risk factors for vision loss, associated costs and quality of life. The examination included measurement of anthropometrics, blood glucose, refraction, ocular biometry, corneal hysteresis, and detailed assessment of the anterior and posterior segments, with photography and optical coherence tomography imaging. Adult participants were invited to donate saliva samples for DNA extraction and storage.\ud \ud Results: The fieldwork was conducted over 13 months in 2013–2014. A representative sample of 10,651 individuals in 3410 households within 120 cluster segments identified 9913 people who were eligible for recruitment.\ud \ud Conclusion: The study methodology was robust and adequate to provide the first population-based estimates of the prevalence and causes of visual impairment and blindness in Trinidad and Tobago. Information was also gathered on risk factors, costs and quality of life associated with vision loss, and on normal ocular parameters for the population aged 40 years and older.

Mayaro virus (MAYV) causes an acute febrile illness similar to that produced by chikungunya virus (CHIKV), an evolutionary relative in the Semliki Forest virus complex of alphaviruses. MAYV emergence is typically sporadic, but recent isolations and outbreaks indicate that the virus remains a public health concern. Given the close phylogenetic and antigenic relationship between CHIKV and MAYV, and widespread distribution of CHIKV, we hypothesized that prior CHIKV immunity may affect MAYV pathogenesis and/or influence its emergence potential. We pre-exposed immunocompetent C57BL/6 and immunocompromised A129 or IFNAR mice to wild-type CHIKV, two CHIKV vaccines, or a live-attenuated MAYV vaccine, and challenged with MAYV. We observed strong cross-protection against MAYV for mice pre-exposed to wild-type CHIKV, and moderately but significantly reduced cross-protection from CHIKV-vaccinated animals. Immunity to other alphavirus or flavivirus controls provided no protection against MAYV disease or viremia. Mechanistic studies suggested that neutralizing antibodies alone can mediate this protection, with T-cells having no significant effect on diminishing disease. Finally, human sera obtained from naturally acquired CHIKV infection cross-neutralized MAYV at high titers in vitro. Altogether, our data suggest that CHIKV infection can confer cross-protective effects against MAYV, and the resultant reduction in viremia may limit the emergence potential of MAYV.

Background Recent emergence of Zika virus (ZIKV), and the global spread of dengue (DENV), chikungunya (CHIKV) and West Nile viruses (WNV) raised urgent need of accurate and affordable molecular diagnosis of these clinically indistinguishable arboviral infections. Objectives We established a pentaplex real-time reverse transcription PCR (rRT-PCR) assay (CII-ArboViroPlex rRT-PCR) for specific and sensitive detection of the African and American genotypes of ZIKV, all four serotypes of DENV, CHIKV, WNV and a housekeeping gene as internal control in single reaction. Study design Specific primers and probe sets were designed for ZIKV, DENV, CHIKV, WNV and RNase P (housekeeping gene) and tested for in-vitro transcribed RNA standards, virus cultures, clinical samples positive for ZIKV, DENV, CHIKV and WNV and limit of detection (LOD) were determined for each. Results Using ten-fold serially diluted in-vitro transcribed RNA, CII- ArboViroPlex rRT-PCR assay has LOD of 100 RNA copies/reaction (Rn) for ZIKV in serum or urine, 100 RNA copies/Rn for DENV in serum, and 10 RNA copies/Rn for CHIKV and WNV in serum. LODs from sera spiked with quantitated viral stocks were 2.6 × 102 GEQ/Rn for ZIKV, 2.2 × 101 GEQ/Rn for DENV-1, 9.4 × 100 GEQ/Rn for DENV-2, 2.3 × 102 GEQ/Rn for DENV-3, 1.4 × 103 GEQ/Rn for DENV-4, 2.7 × 102 GEQ/Rn for CHIKV, and 1.05 × 101 GEQ/Rn for WNV. Conclusions The CII-ArboViroPlex rRT-PCR assay is a quantitative one-step pentaplex rRT-PCR assay for the molecular detection and differential diagnosis of ZIKV, DENV, CHIKV, WNV and a human housekeeping gene control in a single- PCR reaction.

Chewing gum containing xylitol may help prevent caries by reducing levels of mutans streptococci (MS) and lactobacilli in saliva and plaque. Very little is known about other species which are possibly beneficial to oral health. In this study, we employed high-throughput sequencing of the 16S rRNA gene to profile microbial communities of saliva and plaque following short-term consumption of xylitol and sorbitol containing chewing gum. Participants (n = 30) underwent a washout period and were randomly assigned to one of two groups. Each group chewed either xylitol or sorbitol gum for three weeks, before undergoing a second four-week washout period after which they switched to the alternate gum for three weeks. Analysis of samples collected before and after each intervention identified distinct plaque and saliva microbial communities that altered dependent on the order in which gum treatments were given. Neither the xylitol nor sorbitol treatments significantly affected the bacterial composition of plaque. Lactobacilli were undetected and the number of Streptococcus mutans sequence reads was very low and unaffected by either xylitol or sorbitol. However, sorbitol affected several other streptococcal species in saliva including increasing the abundance of S. cristatus, an oral commensal shown to inhibit bacteria associated with chronic periodontitis. Trial registration: ClinicalTrials.gov identifier: NCT03668015.

Background:\ud \ud The conduct of international collaborative genomics research raises distinct ethical challenges that require special consideration, especially if conducted in settings that are research-naïve or resource-limited. Although there is considerable literature on these issues, there is a dearth of literature chronicling approaches taken to address these issues in the field. Additionally no previous ethical guidelines have been developed to support similar research in Trinidad and Tobago.\ud \ud Methods:\ud \ud A literature review was undertaken to identify strategies used to address common ethical issues relevant to human genetics and genomics research in research-naïve or resource-limited settings. Strategies identified were combined with novel approaches to develop a culturally appropriate, multifaceted strategy to address potential challenges in the Genetics Substudy of the National Eye Survey of Trinidad and Tobago (GSNESTT).\ud \ud Results:\ud \ud Regarding the protection of study participants, we report a decision to exclude children as participants; the use of a Community Engagement and Sensitization Strategy to increase the genetic literacy of the target population; the involvement of local expertise to ensure cultural sensitivity and to address potential comprehension barriers in informed consent; and an audit of the informed consent process to ensure valid consent. Concerning the regulation of the research, we report on ethics approvals from relevant authorities; a Materials Transfer Agreement to guide sample ownership and export; and a Sample Governance Committee to oversee data use and data access. Finally regarding the protection of the interests of scientists from the host country, we report on capacity building efforts to ensure that local scientists have access to data collected through the project and appropriate recognition of their contributions in future publications.\ud \ud Conclusion:\ud \ud This paper outlines an ethical framework for the conduct of population-based genetics and genomics research in Trinidad and Tobago; highlights common issues arising in the field and strategies to address these.

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&ndash, 2014 versus 2015&ndash, 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, Artibeus jamaicensis planirostris 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.

Rabies virus is the only Lyssavirus species found in the Americas. In discussions about rabies, Latin America and the Caribbean are often grouped together. Our study aimed to independently analyse the rabies situation in the Caribbean and examine changes in rabies spatiotemporal epidemiology. A questionnaire was administered to the 33 member countries and territories of the Caribbean Animal Health Network (CaribVET) to collect current data, which was collated with a literature review. Rabies was endemic in ten Caribbean localities, with the dog, mongoose, and vampire bat identified as enzootic reservoirs. The majority of animal cases occurred in Puerto Rico, the Dominican Republic, and Haiti, while human cases only consistently occurred in the latter two areas. Rabies vaccination was conducted for high-risk animal populations with variable coverage, and rabies diagnostic capacities varied widely throughout the region. Illegal importation and natural migration of animals may facilitate the introduction of rabies virus variants into virus-na&iuml, ve areas. Passive surveillance, together with enhanced methods and serological screening techniques, can therefore be of value. The insularity of the Caribbean makes it ideal for conducting pilot studies on reservoir host population management. Best practice guidelines developed for these reservoir hosts can be individually modified to the epidemiological status and available resources within each locality.

Fowl adenovirus (FAdV), which causes the high-impact diseases such as inclusion body hepatitis and hepatitis-hydropericardium syndrome, is of major concern to the poultry industry internationally. This study was carried out in direct response to mortality rates of up to 75% in commercial broiler flocks in Trinidad, West Indies. Symptoms in 3- to 8-week-old broilers and 13- to 18-week-old pullets pointed to infection with an immunosuppressive viral pathogen. The objectives of the study were to determine whether the infectious agent FAdV, along with other viral pathogens, was responsible for the clinical disease, and to obtain information on the serotypes of FAdV that were infecting the birds. Tissue samples from clinically affected birds from eight different farms were tested for chicken infectious anaemia virus (CIAV) and infectious bursal disease virus (IBDV) by real-time reverse transcription polymerase chain reaction (PCR) and for FAdV by conventional PCR. The birds tested positive for FAdV and CIAV, but negative for IBDV. The gene corresponding to the L1 loop of the hexon protein for FAdV was amplified and sequenced. Phylogenetic analysis of seven FAdV strains inferred that four serotypes were likely to be circulating in the chickens. Well supported genetic relatedness was observed for serotype 8a (97.8%), 8b (97.8%), 9 (95.8%) and 11 (98.8%-99.5%). This is the first published report from Trinidad and Tobago on the presence and circulation of pathogenic FAdV strains, in combination with CIAV, in poultry. The data demonstrate a possible need for the introduction of serotype-specific vaccines against FAdV, as well as vaccines against CIAV, in broilers in the region and emphasize the importance of maintaining high levels of biosecurity on farms to prevent the spread of these potentially devastating viruses between farms.

Vampire bat-transmitted human rabies was first recognized in Trinidad during a major outbreak during the first half of the 20th century. To date, Trinidad is the only Caribbean island with vampire bat-transmitted rabies. Herein, we summarized the epidemiological situation of rabies in Trinidad during the period 1971–2015 through the analysis of field and laboratory records. During the study period, 259 domestic and wild animal rabies cases were laboratory confirmed with an annual median of 2 animal rabies cases. Over the 45 years, five significant epizootic events occurred (in 1974, 1997–1998, 2000, 2010 and 2012–2013) over which there was a significant increasing trend for the occurrence of rabies cases. The highest number of cases (87 cases) occurred during the 1997–1998 event, and the rabies positive proportion, was highest (0.7, 95% CI 0.52-0.84) for the year 2000. Rabies risk was highest for cattle (negative binomial parameter estimate 4.84, 95% CI 3.45–6.76), although numerous rabies cases were seen in the caprine population during the study period. In light of this finding, consideration should be given to including the small ruminant population in the national rabies vaccination program. Outbreaks affected mainly the counties of St. Patrick and St. George East, with epidemic progression outwards, and these areas should be prioritized for prevention and control efforts.

A total of 2,691 mosquitoes representing 17 species was collected from eight locations in southwest Cameroon and screened for pathogenic viruses. Ten isolates of a novel reovirus (genus Dinovernavirus ) were detected by culturing mosquito pools on Aedes albopictus (C6/36) cell cultures. A virus that caused overt cytopathic effects was isolated, but it did not infect vertebrate cells or produce detectable disease in infant mice after intracerebral inoculation. The virus, tentatively designated Fako virus (FAKV), represents the first 9-segment, double-stranded RNA (dsRNA) virus to be isolated in nature. FAKV appears to have a broad mosquito host range, and its detection in male specimens suggests mosquito-to-mosquito transmission in nature. The structure of the T=1 FAKV virion, determined to subnanometer resolution by cryoelectron microscopy (cryo-EM), showed only four proteins per icosahedral asymmetric unit: a dimer of the major capsid protein, one turret protein, and one clamp protein. While all other turreted reoviruses of known structures have at least two copies of the clamp protein per asymmetric unit, FAKV's clamp protein bound at only one conformer of the major capsid protein. The FAKV capsid architecture and genome organization represent the most simplified reovirus described to date, and phylogenetic analysis suggests that it arose from a more complex ancestor by serial loss-of-function events. IMPORTANCE We describe the detection, genetic, phenotypic, and structural characteristics of a novel Dinovernavirus species isolated from mosquitoes collected in Cameroon. The virus, tentatively designated Fako virus (FAKV), is related to both single-shelled and partially double-shelled viruses. The only other described virus in this genus was isolated from cultured mosquito cells. It was previously unclear whether the phenotypic characteristics of that virus were reflective of this genus in nature or were altered during serial passaging in the chronically infected cell line. FAKV is a naturally occurring single-shelled reovirus with a unique virion architecture that lacks several key structural elements thought to stabilize a single-shelled reovirus virion, suggesting what may be the minimal number of proteins needed to form a viable reovirus particle. FAKV evolved from more complex ancestors by losing a genome segment and several virion proteins.

Highlights • High prevalence of bluetongue virus in Trinidad. • Identification of six bluetongue virus serotypes in Trinidad. • First isolation of BTV-2 and BTV-5 in Trinidad. • Risk of emergence of reassortant viruses., To better understand risks associated with trading cattle, it is important to know which serotypes of Bluetongue virus (BTV) are circulating within the exporting and importing country. Hence, this study was conducted to identify the circulating serotypes of BTV in Trinidad. Blood samples were collected monthly from sixty BTV- naïve imported cattle over a six month period after their arrival in the country. Virological (PCR and virus isolation) and serological (ELISA) analyses were carried out on the samples and CDC light traps were placed near the cattle enclosure to trap and identify the species of Culicoides biting midges that were present. All of the cattle seroconverted for BTV antibodies within three months of their arrival in the country and real-time reverse transcription PCR (rRT-PCR) detected BTV-RNA in the samples throughout the remainder of the study. The patterns of infection observed in the cattle indicated serial infections with multiple serotypes. A combination of BTV serotype-specific rRT-PCR on the original blood samples and virus isolation followed by serotype-specific rRT-PCR on selected samples, confirmed the presence of BTV serotypes 1, 2, 3, 5, 12 and 17. This is the first report of BTV-2 and BTV-5 in Trinidad. Light-suction traps placed in close proximity to the cattle predominantly trapped Culicoides insignis Lutz 1913 species (96%), with a further six Culicoides species making up the remaining 4% of trapped samples. The circulation of multiple BTV serotypes in Trinidad underlines the need for regular surveillance, which will contribute to the development of risk assessments for trade in livestock.

Vampire bat-transmitted rabies was first recognized in Trinidad during a major outbreak reported in 1925. Trinidad is the only Caribbean island with vampire bat-transmitted rabies. We conducted a literature review to describe the changing epidemiology of rabies in Trinidad and give a historical perspective to rabies prevention and control measures on the island. The last human case of rabies occurred in 1937 and although no case of canine-transmitted rabies was reported since 1914, sporadic outbreaks of bat-transmitted rabies still occur in livestock to date. Over the last century, seven notable epidemics were recorded in Trinidad with the loss of over 3000 animals. During the 1950s, several measures were effectively adopted for the prevention and control of the disease which led to a significant reduction in the number of cases. These measures include: vampire bat population control, livestock vaccination, and animal surveillance. However, due to lapses in these measures over the years (e.g., periods of limited vampire control and incomplete herd vaccination), epidemics have occurred. In light of the significant negative impact of rabies on animal production and human health, rabies surveillance in Trinidad should be enhanced and cases evaluated towards the design and implementation of more evidence-based prevention and control programs.

Local transmission of chikungunya virus (CHIKV) was first detected in the Americas in December 2013, after which it spread rapidly throughout the Caribbean islands and American mainland, causing a major chikungunya fever epidemic. Previous phylogenetic analysis of CHIKV from a limited number of countries in the Americas suggests that an Asian genotype strain was responsible, except in Brazil where both Asian and East/Central/South African (ECSA) lineage strains were detected. In this study, we sequenced thirty-three complete CHIKV genomes from viruses isolated in 2014 from fourteen Caribbean islands, the Bahamas and two mainland countries in the Americas. Phylogenetic analyses confirmed that they all belonged to the Asian genotype and clustered together with other Caribbean and mainland sequences isolated during the American outbreak, forming an 'Asian/American' lineage defined by two amino acid substitutions, E2 V368A and 6K L20M, and divided into two well-supported clades. This lineage is estimated to be evolving at a mean rate of 5 x 10-4 substitutions per site per year (95% higher probability density, 2.9-7.9 x 10-4) and to have arisen from an ancestor introduced to the Caribbean (most likely from Oceania) in about March 2013, 9 months prior to the first report of CHIKV in the Americas. Estimation of evolutionary rates for individual gene regions and selection analyses indicate that (in contrast to the Indian Ocean Lineage that emerged from the ECSA genotype followed by adaptive evolution and with a significantly higher substitution rate) the evolutionary dynamics of the Asian/American lineage are very similar to the rest of the Asian genotype and natural selection does not appear to have played a major role in its emergence. However, several codon sites with evidence of positive selection were identified within the non-structural regions of Asian genotype sequences outside of the Asian/American lineage.

Tacaribe virus (TCRV) was isolated in the 1950s from artibeus bats captured on the island of Trinidad. The initial characterization of TCRV suggested that artibeus bats were natural reservoir hosts. However, nearly 60 years later experimental infections of Jamaican fruit bats (Artibeus jamaicensis) resulted in fatal disease or clearance, suggesting artibeus bats may not be a reservoir host. To further evaluate the TCRV reservoir host status of artibeus bats, we captured bats of six species in Trinidad for evidence of infection. Bats of all four fruigivorous species captured had antibodies to TCRV nucleocapsid, whereas none of the insectivore or nectarivore species did. Many flat-faced fruit-eating bats (A. planirostris) and great fruit-eating bats (A. literatus) were seropositive by ELISA and western blot to TCRV nucleocapsid antigen, as were two of four Seba’s fruit bats (Carollia perspicillata) and two of three yellow-shouldered fruit bats (Sturnira lilium). Serum neutralization tests failed to detect neutralizing antibodies to TCRV from these bats. TCRV RNA was not detected in lung tissues or lung homogenates inoculated onto Vero cells. These data indicate that TCRV or a similar arenavirus continues to circulate among fruit bats of Trinidad but there was no evidence of persistent infection, suggesting artibeus bats are not reservoir hosts.

Oryzoborus angolensis (Lesser Seed-Finch), Oryzoborus crassirostris (Large-billed Seed-Finch), and Sporophila intermedia (Grey Seedeater) are finch species native to the Caribbean island of Trinidad. These species are locally trapped and kept for their song, but with declining native populations, enthusiasts have turned to illegally importing birds from the South American mainland. The smuggling of wild birds from South America poses significant disease risks to the native bird species of Trinidad. Herein we describe the first case of poxviral infection in these illegally imported birds in Trinidad and partial genome sequence of the causative agent. Phylogenetic analysis of the 4b core protein sequence indicated that the avian poxvirus identified was most closely related to a 2012 avian pox sequence from Brazil, with 96.2% and 98.1% identity at the nucleotide and amino acid level.

Yellow fever virus (YFV) is historically one of the most important viruses to affect human populations. Despite the existence of highly effective vaccines for over 70 years, yellow fever remains a significant and re-emerging cause of morbidity and mortality in endemic and high-risk regions of South America and Africa. The virus may be maintained in sylvatic enzootic/epizootic, transitional and urban epidemic transmission cycles with geographic variation in terms of levels of genetic diversity, the nature of transmission cycles and patterns of outbreak activity. In this review we consider evolutionary and ecological factors underlying YFV emergence, maintenance and spread, geographic distribution and patterns of epizootic/epidemic activity.

The CD45 antigen is essential for normal antigen receptor-mediated signalling in lymphocytes, and different patterns of splicing of CD45 are associated with distinct functions in lymphocytes. Abnormal CD45 splicing has been recognized in humans, caused by a C77G transversion in the gene encoding CD45 (PTPRC). Recently the C77G polymorphism has been associated with multiple sclerosis and increased susceptibility to HIV-1 infection. These studies suggest that the regulation of CD45 splicing may be critical for the proper function of the immune system. Because of these data we examined the frequency of the C77G allele in African and Asian populations from countries with high or low prevalence of HIV infection. Here we report that the variant CD45 C77G allele is absent in African populations. We further show that populations living in the Pamir mountains of Central Asia have a very high prevalence of the C77G variant.

Seroprevalence rates of selected arboviruses in animal populations in Trinidad were determined using serum samples collected between 2006 and 2009 from horses (n=506), cattle (n=163), sheep (n=198), goats (n=82), pigs (n=184), birds (n=140), rodents (n=116), and other vertebrates (n=23). The sera were screened for antibodies to West Nile virus (WNV), St. Louis encephalitis virus (SLEV), Ilheus virus (ILHV), Bussuquara virus (BSQV), Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV), using hemagglutination inhibition assay (HIA) and epitope-blocking enzyme-linked immunosorbent assays (ELISA). Antibodies to SLEV were detected in a total of 49 (9.7%) horses, 8 (4.9%) cattle, 1 (1.2%) goat, 2 (1.4%) wild birds, and 3 (2.2%) wild rodents by both methods. In contrast, antibodies to EEEV, VEEV, and WNV were detected only in horses, at rates of 4.3%, 0.8%, and 17.2%, respectively, by ELISA, and IgM capture ELISA was WNV-positive in 3 (0.6%) of these sera. Among locally bred unvaccinated horses that had never left Trinidad, seroprevalence rates against WNV were 12.1% and 17.2% by ELISA and HIA, respectively. The presence of WNV- and SLEV-specific antibodies in a representative sample of horse sera that were both ELISA- and HIA-seropositive was confirmed by plaque reduction neutralization testing (PRNT). Antibodies to ILHV and BSQV were not detected in any of the serum samples tested (i.e., sera from horses, other livestock, and wild birds in the case of ILHV, and wild mammals in the case of BSQV). The data indicate the presence of WNV in Trinidad, and continuing low-level circulation of SLEV, EEEV, and VEEV.

Problem-based learning (PBL) and concept mapping have been shown to promote active and meaningful learning.To design a method of PBL that includes concept mapping and examine students' perceptions of this form of PBL.We designed a 5-phase method of PBL which produced three clearly identifiable mapping phases that reflected the learning activities during the tutorial: (1) the initial understanding of the clinical problem, (2) students' prior knowledge of the problem, (3) the final understanding of the problem following self-directed study. The process of developing the second and third phases of the map involved the students answering questions that they generated on two occasions to give the entire process a 5-phase approach. Each student was exposed to both methods of PBL: a conventional 7-step method (Maastricht type) and the modified PBL (5-phase) method. We used a questionnaire to evaluate the students' perceptions of the two methods in four learning domains.The students' ratings for the 5-phase method were significantly higher than for the 7-step method (paired t-test) on all items on the questionnaire.The students perceived the 5-phase method as promoting their passion for learning, and developing their cognitive, metacognitive and interpersonal skills.

Mosquito surveillance was carried out in three forested regions of Trinidad during July 2007–March 2009. A total of 185,397 mosquitoes representing at least 46 species was collected, divided into pools of 1–50 mosquitoes according to species and sex, and screened for arboviruses using cytopathic effect assays on Vero cell monolayers. Eighty-five viruses were isolated, including members of the genera Alphavirus (Mucambo virus; MUCV) and Orthobunyavirus (Caraparu, Oriboca, Bimiti, and Wyeomyia viruses). Species of the Culex subgenus Melanoconion accounted for 56% of the total number of mosquitoes collected and 97% of the viruses isolated; Cx. (Mel.) portesi accounted for 92% of virus isolations. Our results also implicate for the first time Aedes (Ochlerotatus) hortator as a potential vector of MUCV. Phylogenetic analyses of 43 MUCV strains suggest population subdivision within Trinidad, consistent with the hypothesis of enzootic maintenance in localized rodent populations.

A study was conducted to determine the seroprevalence of leptospirosis and infecting serovars across livestock (cattle, sheep, goats, and pigs) in Trinidad using the microscopic agglutination test with an international panel of 23 serovars. Of a total of 590 cattle tested, 21.5% were seropositive with agglutinations to 13 of the 23 antigens used in the panel. Icterohaemorrhagiae (9.3%), Sejroe (4.1%), Ballum (4.1%), and Autumnalis (1.9%) were the predominant serogroups detected in the cattle sampled (n = 590). Of 222 sheep tested, 5.0% were seropositive with agglutinations to five serovars belonging to two serogroups. These serogroups were Autumnalis at 2.7%, and Icterohaemorrhagiae at 2.3% of all sheep tested (n = 222). Of a total of 180 goats tested, 3.3% were seropositive, all agglutinating to the Icterohaemorrhagiae serogroup, 1.7% to serovar Copenhageni, 1.1% to serovar Mankarso, and 0.6% to serovar Icterohaemorrhagiae. Among pigs (n = 200), 5.0% were seropositive for five serovars belonging to three serogroups. These serogroups were Icterohaemorrhagiae at 2.5%, Australis at 2%, and Ballum at 0.5%. Overall, age and sex of animals were not significantly associated with leptospirosis with the exception of cattle where age was a significant factor for seropositivity. It was concluded that for livestock, leptospirosis may be an important zoonotic and economic disease, particularly in the case of cattle. It is imperative that the impact of leptospirosis on abortion, stillbirths, and decreased milk production in livestock in the country be assessed.

A hamster model was used to determine the efficacy of commercially prepared canine vaccines against Leptospira serovars circulating in Trinidad and to assess the effectiveness of killed whole-cell vaccines prepared from local isolates. The local isolates used for vaccine preparation and challenge were isolates of serovars Copenhageni and Mankarso obtained from a local dog and rodent. Their estimated lethal dose-50 (LD(50)) were 5 and 10 organisms, respectively and clinical signs observed on infection were consistent with leptospirosis. An unvaccinated control group of hamsters and other groups of hamsters that had been vaccinated with 3 doses of (i) in-house whole-cell Copenhageni vaccine, (ii) in-house whole-cell Mankarso vaccine, (iii) commercial vaccine Brand A or (iv) commercial vaccines Brand B were challenged with 1000 times the LD(50) of the respective challenge serovar. The most commonly used commercial vaccine (Brand A) did not offer protection to challenged hamsters, whereas Brand B facilitated the renal carrier state of the Leptospira organism. In contrast the whole-cell vaccines developed from local strains of serovars Copenhageni and Mankarso, protected all hamsters tested from both clinical disease and renal carrier states.

In the 1950s and 1960s, alphaviruses in the Venezuelan equine encephalitis (VEE) antigenic complex were the most frequently isolated arboviruses in Trinidad. Since then, there has been very little research performed with these viruses. Herein, we report on the isolation, sequencing, and phylogenetic analyses of Mucambo virus (MUCV; VEE complex subtype IIIA), including 6 recently isolated from Culex (Melanoconion) portesi mosquitoes and 11 previously isolated in Trinidad and Brazil. Results show that nucleotide and amino acid identities across the complete structural polyprotein for the MUCV isolates were 96.6–100% and 98.7–100%, respectively, and the phylogenetic tree inferred for MUCV was highly geographically- and temporally-structured. Bayesian analyses suggest that the sampled MUCV lineages have a recent common ancestry of approximately 198 years (with a 95% highest posterior density (HPD) interval of 63–448 years) prior to 2007, and an overall rate of evolution of 1.28 × 10− 4 substitutions/site/yr.

Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.

Interactions of killer cell immunoglobulin-like receptors (KIRs) with major histocompatibility complex (MHC) class I ligands diversify natural killer cell responses to infection. By analyzing sequence variation in diverse human populations, we show that the KIR3DL1/S1 locus encodes two lineages of polymorphic inhibitory KIR3DL1 allotypes that recognize Bw4 epitopes of protein">HLA-A and HLA-B and one lineage of conserved activating KIR3DS1 allotypes, also implicated in Bw4 recognition. Balancing selection has maintained these three lineages for over 3 million years. Variation was selected at D1 and D2 domain residues that contact HLA class I and at two sites on D0, the domain that enhances the binding of KIR3D to HLA class I. HLA-B variants that gained Bw4 through interallelic microconversion are also products of selection. A worldwide comparison uncovers unusual KIR3DL1/S1 evolution in modern sub-Saharan Africans. Balancing selection is weak and confined to D0, KIR3DS1 is rare and KIR3DL1 allotypes with similar binding sites predominate. Natural killer cells express the dominant KIR3DL1 at a high frequency and with high surface density, providing strong responses to cells perturbed in Bw4 expression.

Local transmission of Chikungunya virus (CHIKV) was first documented in Trinidad and Tobago (T&T) in July 2014 preceding a large epidemic. At initial presentation, it is difficult to distinguish chikungunya fever (CHIKF) from other acute undifferentiated febrile illnesses (AUFIs), including life-threatening dengue disease. We characterised and compared dengue virus (DENV) and CHIKV infections in 158 patients presenting with suspected dengue fever (DF) and CHIKF at a major hospital in T&T, and performed phylogenetic analyses on CHIKV genomic sequences recovered from 8 individuals. The characteristics of patients with and without PCR-confirmed CHIKV were compared using Pearson’s χ2 and student’s t-tests, and adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were determined using logistic regression. We then compared signs and symptoms of people with RT-qPCR-confirmed CHIKV and DENV infections using the Mann-Whitney U, Pearson’s χ2 and Fisher’s exact tests. Among the 158 persons there were 8 (6%) RT-qPCR-confirmed DENV and 30 (22%) RT-qPCR-confirmed CHIKV infections. Phylogenetic analyses showed that the CHIKV strains belonged to the Asian genotype and were most closely related to a British Virgin Islands strain isolated at the beginning of the 2013/14 outbreak in the Americas. Compared to persons who were RT-qPCR-negative for CHIKV, RT-qPCR-positive individuals were significantly more likely to have joint pain (aOR: 4.52 [95% CI: 1.28–16.00]), less likely to be interviewed at a later stage of illness (days post onset of fever—aOR: 0.56 [0.40–0.78]) and had a lower white blood cell count (aOR: 0.83 [0.71–0.96]). Among the 38 patients with RT-qPCR-confirmed CHIKV or DENV, there were no significant differences in symptomatic presentation. However when individuals with serological evidence of recent DENV or CHIKV infection were included in the analyses, there were key differences in clinical presentation between CHIKF and other AUFIs including DF, which can be used to triage patients for appropriate care in the clinical setting., Author Summary Chikungunya virus (CHIKV) recently emerged in the Americas and caused a major epidemic of chikungunya fever (CHIKF). While not usually life threatening, CHIKF is a debilitating and often chronic illness resulting in major morbidity and economic losses. It is difficult to distinguish CHIKF from other viral illnesses that cause acute fevers including dengue fever (DF), an important consideration since DF can be life-threatening and early identification and treatment of cases is key to reducing mortality. In this study we investigated individuals presenting to a major hospital in Trinidad and Tobago (T&T) with suspected DF or CHIKF, and identified signs and symptoms that distinguish these two illnesses. We also recovered complete genome sequences for CHIKV and show that the etiologic strain in T&T is a closely related descendent of the strain first isolated from the British Virgin Islands at the beginning of the outbreak in the Americas.

Using DNA extracted from 112 parasitised blood blots, we screened for the population marker of chloroquine resistance (CQR) pfcrt K76T in Plasmodium falciparum infections from Guyana. Pfmdr1 mutations S1034C, N1042D, and D1246Y also associated with CQR were surveyed as well in 15 isolates for which the in vitro responses to CQ were known. Results indicate that the pfcrt K76T is ubiquitous in this environment, and confirmatory sequencing of codons 72 and 76 revealed two novel allelic sequences SVMIT and RVMNT in addition to the previously identified CVMNT and SVMNT haplotypes. The frequency of the pfcrt K76T despite its presence in both CQR and CQS (chloroquine sensitive) infections measured in vivo and in vitro, suggests that it is a useful population marker in this low-transmission setting of sweeping CQR.

We sequenced the envelope (E) genes of 59 DEN-2 isolates collected from ten Caribbean islands, six South American countries, and two Central American countries between 1981 and 2000, a period characterized by hyperendemicity and increased incidence of severe dengue. Fifty-two isolates belonged to “American/Asian” subtype IIIb, possessing a characteristic polar residue at envelope aa position 390 (N [n = 48] or S [n = 4]) common to that group. Six isolates from Trinidad (1981), Honduras (1991 [4]), and El Salvador (1987) fell into the “Native American” subtype V (D at aa 390), and one from Honduras (1986) belonged to “Asian” subtype I. The data suggest that after its first isolation in the Caribbean in 1981, genotype IIIb spread throughout the Americas and effectively replaced subtype V throughout the Caribbean basin. The strain also evolved into several distinct lineages, based on substitutions in the E glycoprotein (amino acids 91 and 131), two of which were still in circulation in 2000. Interestingly, a molecular clock did not fit the data well, suggesting that other sources of rate variation, such as differential selection or differences in effective population sizes, may exist among lineages. Our results indicate the importance of large temporal- and geographical-scale phylogenetic studies in understanding disease dynamics, particularly where replacements between regions can occur.

Analysis of FcgammaRIIA alleles in Pakistanis and in Trinidadians of South Asian, African and mixed ancestry revealed no significant differences between Trinidadian South Asians and Pakistanis. H131 homozygotes were more common among Trinidadian South Asians than among Africans and those of mixed ancestry. Comparison with other populations revealed east-west geographic gradients of allele frequencies.

We sequenced the E gene and adjacent prM/M and NS1 junctions (1940 bp) of 48 Dengue-4 (DEN-4) isolates collected between 1981 and 1999 from 8 Caribbean islands and from 7 South and Central American countries. Phylogenetic analysis confirms a single introduction in the early 1980s and a high degree of gene flow resulting in a pattern of evolution defined more by time period than geographic origin, especially within the Caribbean basin. A modern Caribbean clade consisting of four distinct lineages has arisen, comprised of isolates from Caribbean islands and nearby regions of South America. This clade is defined by three amino acid substitutions in the E (aa 163 and 351) and NS1 (aa 52) proteins. These findings highlight the importance of migration and gene flow in dengue viral change and suggest that efforts to understand disease dynamics in the Caribbean basin need to focus at regional, rather than local scales.

Natural killer (NK) and some T cells express killer cell immunoglobulin-like receptors (KIRs), which interact with HLA class I expressed by target cells and consequently regulate cytolytic activity. The number of KIR loci can vary and so a range of genetic profiles is observed. We have determined the KIR genetic profiles from one African (n = 62) and two South Asian (n = 108, n = 78) populations. Several of the KIRs are present at significantly different frequencies between the two major ethnic groups (eg KIR2DS4 gene frequency 0.82 African, 0.47 S Asian. Pc < 1 × 10−6) and this is due to uneven distribution of two KIR haplotype families ‘A’ and ‘B’. All three populations described here displayed a greater degree of diversity of KIR genetic profiles than other populations investigated, which indicates further complexity of underlying haplotypes; in this respect we describe two individuals who appear homozygous for a large deletion including the previously ubiquitous 2DL4. We have also reanalysed three populations that we studied previously, for the presence of a KIR which is now known to be an indicator of the ‘B’ haplotype. South Asians had the highest overall frequencies of all KIR loci characteristic of ‘B’ haplotypes (Pc < 0.0001 to < 0.004). Furthermore, gene frequency independent deviances in the linkage disequilibrium were apparent between populations.