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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.

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., (© 2019 Blackwell Verlag GmbH.)

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)., (Copyright © 2018. Published by Elsevier B.V.)

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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance are limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time reverse transcriptase-PCR (RT-PCR) detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; P=0.01)., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A serosurvey of antibodies against selected flaviviruses and alphaviruses in 384 bats (representing 10 genera and 14 species) was conducted in the Caribbean island of Trinidad. Sera were analysed using epitope-blocking enzyme-linked immunosorbent assays (ELISAs) specific for antibodies against West Nile virus (WNV), Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), all of which are zoonotic viruses of public health significance in the region. Overall, the ELISAs resulted in the detection of VEEV-specific antibodies in 11 (2.9%) of 384 bats. Antibodies to WNV and EEEV were not detected in any sera. Of the 384 sera, 308 were also screened using hemagglutination inhibition assay (HIA) for antibodies to the aforementioned viruses as well as St. Louis encephalitis virus (SLEV; which also causes epidemic disease in humans), Rio Bravo virus (RBV), Tamana bat virus (TABV) and western equine encephalitis virus (WEEV). Using this approach, antibodies to TABV and RBV were detected in 47 (15.3%) and 3 (1.0%) bats, respectively. HIA results also suggest the presence of antibodies to an undetermined flavivirus(es) in 8 (2.6%) bats. Seropositivity for TABV was significantly (P<0.05; χ2) associated with bat species, location and feeding preference, and for VEEV with roost type and location. Differences in prevalence rates between urban and rural locations were statistically significant (P<0.05; χ2) for TABV only. None of the aforementioned factors was significantly associated with RBV seropositivity rates., (© 2014 Blackwell Verlag GmbH.)

Unlabelled: 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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Stray dogs (n=207), suspected canine cases of leptospirosis (n=50) and rats (n=200) from the Caribbean island of Trinidad were subjected to the Microscopic Agglutination Test (MAT) for leptospirosis. The seroprevalence in stray dogs was 15.5% (n=32), the predominant serogroup was Icterohaemorrhagiae (14.5%; n=30) with agglutinations to serovars Copenhageni at 5.8%, Icterohaemorrhagiae at 4.8%, Mankarso at 3.9%. The seroprevalence among suspected canine cases was 72% (n=36) with Icterohaemorrhagiae again being the predominant serogroup at 60% inclusive of serovars: Copenhageni, 44%; Mankarso, 14%; and Icterohaemorrhagiae 2%. A seroprevalence of 16.5% was determined in rats, all agglutinations were to the Icterohaemorrhagiae serogroup (inclusive of serovars Copenhageni, 9.5%; Icterohaemorrhagiae, 5.5%; and Mankarso, 1.5%). Overall serovar Copenhageni was the most common serovar as 11.6% of all the animal species tested by the MAT were positive and may be an important zoonotic serovar in Trinidad. The titres of infecting serovars of Leptospira in suspected canine cases of leptospirosis were considerably higher than that found in stray dogs and in rats where the lowest titres were found. Age and sex were not significant risk factors except in the case of rats where age was significant, indicating that juvenile rats were at a significantly higher risk. There was no definite pattern of the distribution of positive animals or the serovars when using the MAT. Data obtained in the current study indicate that dogs and rats in Trinidad have the potential to be sources of leptospiral infections for humans. This potential has public health implications making it imperative to control rat and stray dog populations in the island to reduce the risk of human leptospirosis.

The Colletotrichum gloeosporioides species complex is among the most destructive fungal plant pathogens in the world, however, identification of isolates of quarantine importance to the intra-specific level is confounded by a number of factors that affect phylogenetic reconstruction. Information bias and quality parameters were investigated to determine whether nucleotide sequence alignments and phylogenetic trees accurately reflect the genetic diversity and phylogenetic relatedness of individuals. Sequence exploration of GAPDH, ACT, TUB2 and ITS markers indicated that the query sequences had different patterns of nucleotide substitution but were without evidence of base substitution saturation. Regions of high entropy were much more dispersed in the ACT and GAPDH marker alignments than for the ITS and TUB2 markers. A discernible bimodal gap in the genetic distance frequency histograms was produced for the ACT and GAPDH markers which indicated successful separation of intra- and inter-specific sequences in the data set. Overall, analyses indicated clear differences in the ability of these markers to phylogenetically separate individuals to the intra-specific level which coincided with information bias.

Bat rabies is an emerging disease of public health significance in the Americas. The Caribbean island of Trinidad experiences periodic outbreaks within the livestock population. We performed molecular characterisation of Trinidad rabies virus (RABV) and used a Bayesian phylogeographic approach to investigate the extent to which outbreaks are a result of in situ evolution versus importation of virus from the nearby South American mainland. Trinidadian RABV sequences were confirmed as bat variant and clustered with Desmodus rotundus (vampire bat) related sequences. They fell into two largely temporally defined lineages designated Trinidad I and II. The Trinidad I lineage which included sequences from 1997-2000 (all but two of which were from the northeast of the island) was most closely related to RABV from Ecuador (2005, 2007), French Guiana (1990) and Venezuela (1993, 1994). Trinidad II comprised sequences from the southwest of the island, which clustered into two groups: Trinidad IIa, which included one sequence each from 2000 and 2007, and Trinidad IIb including all 2010 sequences. The Trinidad II sequences were most closely related to sequences from Brazil (1999, 2004) and Uruguay (2007, 2008). Phylogeographic analyses support three separate RABV introductions from the mainland from which each of the three Trinidadian lineages arose. The estimated dates for the introductions and subsequent lineage expansions suggest periods of in situ evolution within Trinidad following each introduction. These data also indicate co-circulation of Trinidad lineage I and IIa during 2000. In light of these findings and the likely vampire bat origin of Trinidadian RABV, further studies should be conducted to investigate the relationship between RABV spatiotemporal dynamics and vampire bat population ecology, in particular any movement between the mainland and Trinidad.

Background: C. gloeosporioides sensu lato is one of the most economically important post-harvest diseases affecting papaya production worldwide. There is currently no information concerning the genetic structure or demographic history of this pathogen in any of the affected countries. Knowledge of molecular demographic parameters for different populations will improve our understanding of the biogeographic history as well as the evolutionary and adaptive potential of these pathogens. In this study, sequence data for ACT, GPDH, β-TUB and ITS gene regions were analyzed for C. gloeosporioides sensu lato and C. truncatum isolates infecting papaya in Trinidad and Mexico in order to determine the genetic structure and demographic history of these populations., Results: The data indicated that Mexico is the ancestral C. gloeosporioides sensu lato population with asymmetrical migration to Trinidad. Mexico also had the larger effective population size but, both Mexico and Trinidad populations exhibited population expansion. Mexico also had greater nucleotide diversity and high levels of diversity for each gene. There was significant sub-division of the Trinidad and Mexico populations and low levels of genetic divergence among populations for three of the four gene regions; β-TUB was shown to be under positive selection. There were also dissimilar haplotype characteristics for both populations. Mutation may play a role in shaping the population structure of C. gloeosporioides sensu lato isolates from Trinidad and from Mexico, especially with respect to the ACT and GPDH gene regions. There was no evidence of gene flow between the C. truncatum populations and it is possible that the Mexico and Trinidad populations emerged independently of each other., Conclusions: The study revealed relevant information based on the genetic structure as well as the demographic history of two fungal pathogens infecting papaya, C. gloeosporioides sensu lato and C. truncatum, in Trinidad and Mexico. Understanding the genetic structure of pathogen populations will assist in determining the evolutionary potential of the pathogen and in identifying which evolutionary forces may have the greatest impact on durability of resistance. Intervention strategies that target these evolutionary forces would prove to be the most practical.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Changes in Dengue virus (DENV) disease patterns in the Americas over recent decades have been attributed, at least in part, to repeated introduction of DENV strains from other regions, resulting in a shift from hypoendemicity to hyperendemicity. Using newly sequenced DENV-1 and DENV-3 envelope (E) gene isolates from 11 Caribbean countries, along with sequences available on GenBank, we sought to document the population genetic and spatiotemporal transmission histories of the four main invading DENV genotypes within the Americas and investigate factors that influence the rate and intensity of DENV transmission. For all genotypes, there was an initial invasion phase characterized by rapid increases in genetic diversity, which coincided with the first confirmed cases of each genotype in the region. Rapid geographic dispersal occurred upon each genotype's introduction, after which individual lineages were locally maintained, and gene flow was primarily observed among neighboring and nearby countries. There were, however, centers of viral diversity (Barbados, Puerto Rico, Colombia, Suriname, Venezuela, and Brazil) that were repeatedly involved in gene flow with more distant locations. For DENV-1 and DENV-2, we found that a "distance-informed" model, which posits that the intensity of virus movement between locations is inversely proportional to the distance between them, provided a better fit than a model assuming equal rates of movement between all pairs of countries. However, for DENV-3 and DENV-4, the more stochastic "equal rates" model was preferred.

Objective: Human papillomavirus (HPV) genotypes and their relative prevalences were determined in a cohort of 310 sexually active women in Trinidad, West Indies., Methods: Cervical samples were collected with Ayre's spatulas and endocervical brushes. Samples were used for the conventional Papanicolaou test and for determining HPV genotypes by amplification of a section of the viral L1 gene, followed by DNA sequencing and probe hybridization., Results: HPV infections were identified in 126 of 310 (40.6%) women. Of them, 83 (65.8%) were infected with high-risk HPV, 16 (12.7%) with low-risk HPV, and 27 (21.4%) with HPV types of unknown risk. HPV 52 (12.7%) was the most frequently occurring high-risk type, followed by HPV 66 (10.3%), HPV 16 (9.5%), and HPV 18 (8.6%). High-risk types HPV 16 and HPV 66 were each found in 3 (20.0%) and HPV 18 was found in 1 (6.6%) of the 15 women with abnormal cytology., Conclusions: Cervical HPV prevalence and heterogeneity of HPV genotypes are high in this Trinidad cohort. The relative importance of HPV genotypes in the development of cervical lesions needs further investigation in Trinidad in order to better understand the epidemiology of HPV infections as well as to determine the role of HPV testing in the screening, prevention, and control of cervical cancer. This pilot study provided important information on the prevalence of HPV genotypes, which will be used in future nationwide studies.

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.

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.

Trinidad, like many other American regions, experiences repeated epizootics of yellow fever virus (YFV). However, it is unclear whether these result from in situ evolution (enzootic maintenance) or regular reintroduction of YFV from the South American mainland. To discriminate between these hypotheses, we carried out a Bayesian phylogeographic analysis of over 100 prM/E gene sequences sampled from 8 South American countries. These included newly sequenced isolates from the recent 2008-2009 Trinidad epizootic and isolates derived from mainland countries within the last decade. The results indicate that the most recent common ancestor of the 2008-2009 epizootic existed in Trinidad 4.2 years prior to 2009 (95% highest probability density [HPD], 0.5 to 9.0 years). Our data also suggest a Trinidad origin for the progenitor of the 1995 Trinidad epizootic and support in situ evolution of YFV between the 1979 and 1988-1989 Trinidad epizootics. Using the same phylogeographic approach, we also inferred the historical spread of YFV in the Americas. The results suggest a Brazilian origin for YFV in the Americas and an overall dispersal rate of 182 km/year (95% HPD, 52 to 462 km/year), with Brazil as the major source population for surrounding countries. There is also strong statistical support for epidemiological links between four Brazilian regions and other countries. In contrast, while there were well-supported epidemiological links within Peru, the only statistically supported external link was a relatively weak link with neighboring Bolivia. Lastly, we performed a complete analysis of the genome of a newly sequenced Trinidad 2009 isolate, the first complete genome for a genotype I YFV isolate.

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., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We determined the frequency of isolation of Leptospira from dogs and rodents, the serovars of Leptospira, and the clinical, gross and histological manifestations in dogs with leptospirosis in Trinidad. From dogs, samples of urine, blood and kidney were collected while only kidney and blood samples of trapped rodents were used. Isolates were cultured and serotyped using a panel of 23 international serovars and monoclonal antibodies. The risk factors for leptospirosis were also determined in owned dogs using a standard questionnaire. Of a total of 468 animals investigated for Leptospira, 70 (15.0%) were positive, comprising nine (18.0%) of 50 suspected canine leptospirosis cases, seven (3.4%) of 207 stray dogs and 54 (25.6%) of 211 rodents. The observation that rodents have a statistically (P<0.05, chi2) higher frequency of isolation emphasizes the importance of rodents as reservoirs of leptospirosis in the country. Copenhageni was the predominant serovar found in 100.0% (7/7), 33.3% (2/6) and 68.5% (37/54) of isolates from suspected canine leptospirosis cases, stray dogs and rodents, respectively. Serovars Icterohaemorrhagiae and Canicola, the two serovars present in the commercial vaccines used locally, were detected in one (1.5%) and zero (0.0%) isolates respectively of the 67 tested. Data provided suggest that the apparent vaccine failure may be a consequence of the fact that the predominant serovar (Copenhageni) detected in sick, apparently healthy dogs and in rodents is not contained in the vaccines used locally to protect dogs against canine leptospirosis.

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 x 10(-4) substitutions/site/yr.

Using a Bayesian coalescent approach on a dataset of 73 envelope gene sequences we estimated substitution rates and dates of divergence for St. Louis encephalitis virus (SLEV) in the Americas. We found significant rate heterogeneity among lineages, such that "relaxed" molecular clock models were much better supported than a strict molecular clock. The mean substitution rate estimated for all SLEV was 4.1x10(-4)substitutions/site/year (95% HPD 2.5-5.7)-higher than previous estimates that relied on the less well-suited strict clock. Mean substitution rates for individual lineages varied from 3.7x10(-4) to 7.2x10(-4)substitutions/site/year. For the first time we also assessed the magnitude and direction of viral gene flow within the Americas. The overall direction of gene flow during the period represented by the phylogeny is from South to North, and the region between 15 degrees N and 30 degrees N latitude appears to be the major source of virus for the rest of North America, which is consistent with migratory birds returning to their northern breeding grounds having acquired infection while wintering in the region of the Gulf of Mexico.

Natural killer (NK) cells contribute to the essential functions of innate immunity and reproduction. Various genes encode NK cell receptors that recognize the major histocompatibility complex (MHC) Class I molecules expressed by other cells. For primate NK cells, the killer-cell immunoglobulin-like receptors (KIR) are a variable and rapidly evolving family of MHC Class I receptors. Studied here is KIR3DL1/S1, which encodes receptors for highly polymorphic human HLA-A and -B and comprises three ancient allelic lineages that have been preserved by balancing selection throughout human evolution. While the 3DS1 lineage of activating receptors has been conserved, the two 3DL1 lineages of inhibitory receptors were diversified through inter-lineage recombination with each other and with 3DS1. Prominent targets for recombination were D0-domain polymorphisms, which modulate enhancer function, and dimorphism at position 283 in the D2 domain, which influences inhibitory function. In African populations, unequal crossing over between the 3DL1 and 3DL2 genes produced a deleted KIR haplotype in which the telomeric "half" was reduced to a single fusion gene with functional properties distinct from its 3DL1 and 3DL2 parents. Conversely, in Eurasian populations, duplication of the KIR3DL1/S1 locus by unequal crossing over has enabled individuals to carry and express alleles of all three KIR3DL1/S1 lineages. These results demonstrate how meiotic recombination combines with an ancient, preserved diversity to create new KIR phenotypes upon which natural selection acts. A consequence of such recombination is to blur the distinction between alleles and loci in the rapidly evolving human KIR gene family.

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.

Bat coronaviruses (Bt-CoVs) are thought to be the precursors of severe acute respiratory syndrome coronavirus. We detected Bt-CoVs in 2 bat species from Trinidad. Phylogenetic analysis of the RNA-dependent RNA polymerase gene and helicase confirmed them as group 1 coronaviruses.

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.

Study Objective: The aim of this retrospective study was to determine the incidence of vinca alkaloid extravasation following administration via syringes or mini-bags., Methods: An electronic survey was sent to pharmacy departments at hospitals throughout Australia. The survey was designed to collect data on the administration methods of vinca alkaloids (vincristine, vinblastine, vinorelbine) and the corresponding documented incidence of extravasation. The questionnaire requested the following information: how the drugs were prepared for administration (syringe or mini-bag); volume of each product; number of items supplied; and the number of cases of extravasation reported for each product., Results: Questionnaires were sent to 228 hospital pharmacy departments in Australia. Sixty-eight questionnaires were received (29.8%), including responses from most major cancer centres. Data represented a range of 3 to 120 months (mean: 38 months) of clinical experience. The reported incidence of vincristine extravasation from syringes was 0.03% (11/37 084) and 0.041% (3/7255) with mini-bags. One case of vinblastine extravasation was reported with syringes 0.013% (1/7913), none from mini-bags (0/1421). Vinorelbine data were difficult to interpret as cases may represent phlebitis rather than extravasation. The reported incidence from vinorelbine syringes was 0.029% (2/6914) and 0.146% (8/5475) with mini-bags. Excluding vinorelbine data, the reported vinca alkaloid extravasation episodes from syringes (0.027%) and mini-bags (0.035%) were found to be similar and infrequent., Conclusion: The data suggest that vinca alkaloids can be given safely as low volume, short infusions via mini-bags. Policies and practices that ensure the same careful monitoring of infusional therapy as is recommended with administration by syringe may further reduce the incidence of untoward effects. Mini-bags should be used for the administration of vinca alkaloids and this practice will prevent the inadvertent intrathecal administration of vinca alkaloids via syringes.

Dengue virus type 4 (DENV-4) was first reported in the Americas in 1981, where it caused epidemics of dengue fever throughout the region. In the same year, the region's first epidemic of dengue hemorrhagic fever was reported, caused by an Asian strain of dengue virus type 2 (DENV-2) that was distinct from the American subtype circulating previously. Despite the importance of these epidemics, little is known about the rates or determinants of viral spread among island and mainland populations or their directions of movement. We employed a Bayesian coalescent approach to investigate the transmission histories of DENV-2 and DENV-4 since their introduction in 1981 and a parsimony method to assess patterns of strain migration. For both viruses there was an initial invasion phase characterized by an exponential increase in the number of DENV lineages, after which levels of genetic diversity remained constant despite reported fluctuations in DENV-2 and DENV-4 activity. Strikingly, viral lineage numbers increased far more rapidly for DENV-4 than DENV-2, indicative of a more rapid rate of exponential population growth in DENV-4 or a higher rate of geographic dispersal, allowing this virus to move more effectively among localities. We propose that these contrasting dynamics may reflect underlying differences in patterns of host immunity. Despite continued gene flow along particular transmission routes, the overall extent of viral traffic was less than expected under panmixis. Hence, DENV in the Americas has a clear geographic structure that maintains viral diversity between outbreaks.

The human leukocyte receptor complex (LRC) of Chromosome 19q13.4 encodes polymorphic and highly homologous genes that are expressed by cells of the immune system and regulate their function. There is an enormous diversity at the LRC, most particularly the variable number of killer cell immunoglobulin-like receptor (KIR) genes. KIR have been associated with several disease processes due to their interaction with polymorphic human leukocyte antigen class I molecules. We have assessed haplotype compositions, linkage disequilibrium patterns and allele frequencies in two Caucasoid population samples (n=54, n=100), using a composite of single-nucleotide polymorphism (SNP) markers and high-resolution, allele-specific molecular genotyping. Particular KIR loci segregated with SNP and other markers, forming two blocks that were separated by a region with a greater history of recombination. The KIR haplotype composition and allele frequency distributions were consistent with KIR having been subject to balancing selection (Watterson's F: P=0.001). In contrast, there was a high inter-population heterogeneity measure for the LRC-encoded leukocyte immunoglobulin-like receptor A3 (LILRA3), indicating pathogen-driven disruptive selection (Wright's FST=0.32). An assessment of seven populations representative of African, Asian and Caucasoid ethnic groups (total n=593) provided little evidence for long-range LRC haplotypes. The different natural selection pressures acting on each locus may have contributed to a lack of linkage disequilibrium between them.

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.

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.

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.

Using polymerase chain reaction-sequence-specific primer (PCR-SSP) typing, this study determined the frequency of human leukocyte antigen (HLA) DR- and -DQ alleles and haplotypes in individuals of African (n = 75), South Asian (n = 98), and mixed (n = 102) ancestry from the Caribbean island of Trinidad. We detected 19 different haplotypes containing DRB3, 8 containing DRB4, 6 containing DRB5, and 6 different haplotypes without DRB3/4/5 genes. Twenty-nine haplotypes were identified in Africans, 24 in the South Asians, and 32 in the mixed group. We detected significant differences between the populations, principally at the DQA1 and DQB1 loci, although the allele frequency for DRB1*0901 was highest in the Africans (p(c) < 0.05). Trinidad African and mixed groups were generally more diverse than the South Asians and displayed a wider range of DRB1-DQB1 associations; DQB1*02 and DQB1*0301 each associated with five to six different DRB1 alleles in the Africans and mixed group but only two in South Asians. In the Africans and the mixed group, DQB1*04 was found with DRB1*0302 and DRB1*04, but only with DRB1*08 in the South Asians. Trinidad Africans revealed consistencies with populations in Western, Central, and Northern Africa, but differed considerably from individual populations on the African continent. Trinidad South Asians displayed similar allele frequencies and associations to other populations from Northern India.

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 x 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.

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.

Trinidadians of South Asian origin have a high prevalence of cardiovascular disease and diabetes compared to Trinidadians of African origin. The degree to which these differences are related to genetic and/or environmental factors is unclear. To determine whether there might be a genetic basis for this difference in prevalence of deleterious phenotypes we examined allele frequencies for candidate genes in atherosclerosis and diabetes. We genotyped 81 consecutive neonates of African origin and 103 consecutive neonates of South Asian origin. We evaluated common polymorphisms in 11 candidate genes for atherosclerosis and diabetes. We found differences between the two subpopulations in the allele frequencies of several candidate genes, including APOE, LIPC, APOC3, PON1, PON2, and PPP1R3. However, the differences in the allele frequencies were not all consistent with the pattern of CHD expression between these two ethnic groups in adulthood. Thus, differences in genetic architecture alone may not explain the wide disparities in disease prevalence between these two subpopulations. It is very likely that environmental factors, or unmeasured genetic factors, influence the genetic susceptibility to disease in these subpopulations.

Pancreatic lipase (EC 3.1.1.3) is an exocrine secretion that hydrolyzes dietary triglycerides in the small intestine. We developed genomic amplification primers to sequence the 13 exons of PNLIP, which encodes pancreatic lipase, in order to screen for possible mutations in cell lines of four children with pancreatic lipase deficiency (OMIM 246600). We found no missense or nonsense mutations in these samples, but we found three silent single-nucleotide polymorphisms (SNPs), namely, 96A/C in exon 3, 486C/T in exon 6, and 1359C/T in exon 13. In 50 normolipidemic Caucasians, the PNLIP 96C and 486T alleles had frequencies of 0.083 and 0.150, respectively. The PNLIP 1359T allele was absent from Caucasian, Chinese, South Asian, and North American aboriginal samples, but had a frequency of 0.085 in an African sample, suggesting that it is a population-specific variant. In an association analysis of 185 African neonates, the PNLIP 1359C/T SNP genotype was significantly associated with concentrations of plasma lipoproteins. These associations were most likely due to linkage disequilibrium with another functional variant at or near PNLIP. Thus, we report three new SNPs for the PNLIP, which may serve as markers for association analyses and for pharmacogenetic studies of pancreatic lipase inhibitors.

We are investigating the binding of a series of monoclonal antibodies to native and detergent-treated human T-lymphotropic virus I (HTLV-I) envelope proteins to explore their conformation. A comparison of our data with previously published findings suggests that a central neutralization domain (aa 175-200) is folded such that only short stretches are exposed at the surface of the native envelope protein complex. However, the complete domain becomes accessible after treatment with mild non-ionic detergents, suggesting that envelope subunit interaction may partially obscure this domain. We further provide immunochemical evidence that a region containing a heptad repeat in the extracellular part of the transmembrane protein is folded towards the interior of the HTLV-I envelope complex.

The HTLV-I producer cell line C10/MJ2 does not induce syncytium formation with HTLV-I receptor expressing cells. Here we show that this cell line produces a truncated envelope protein, which, because of a premature stop codon, lacks the hydrophobic membrane anchor domain of the transmembrane protein (TM). Despite lacking a membrane anchor this envelope protein is expressed on the cell surface and associated with released virions. However, its incorporation into virions seems less efficient than that of a full-length envelope glycoprotein and some of its released into the cell culture supernatant as soluble surface glycoprotein (SU)-TM complexes. Small amounts of such a truncated envelope glycoprotein were also found in the fusion-competent HTLV-I producer cell line MT2. Premature truncation of HTLV-I envelope proteins in producer cell lines may result from in vitro selection for a less fusogenic phenotype. The association of truncated HTLV-I envelope proteins with virions and cell surfaces may reflect interactions between the SU domain and cellular membranes, possibly with the cellular receptor for HTLV-I.

We sequenced the envelope genes of Human T-cell leukemia type I viruses (HTLV-I) derived from five Brazilian, two Caribbean, and one Romanian case of adult T-cell leukemia after amplification of the complete env gene by PCR. A comparison with previously reported HTLV-I sequences revealed that, although highly homologous, no two env sequences were identical. All envelope sequences differed from each other by 0.3-2.1% nucleotide differences. The five Brazilian sequences clustered together and were about as different from each other (0.5-0.75% nucleotide difference) as were three previously reported Japanese sequences (0.7-0.95%). In contrast, sequences of Caribbean origin were less homogeneous (0.5-1.9% nucleotide differences within this group). The Romanian sequence was not significantly more divergent than any of the others and was closest to our two Caribbean sequences. We observed two changes in a region (aa 176-209) which has previously been shown to contain a linear antibody epitope recognized by most human sera from seropositive individuals. One of these changes affects the binding of monoclonal antibodies to this epitope demonstrating the variability of an antibody epitope in the HTLV-I envelope.