Bluetongue virus infection in naïve cattle: Identification of circulating serotypes and associated Culicoides biting midge species in Trinidad.

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.

Understanding the evolution and spread of chikungunya virus in the Americas using complete genome sequences.

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 × 10-4 substitutions per site per year (95% higher probability density, 2.9-7.9 × 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.

Seroepidemiology of selected alphaviruses and flaviviruses in bats in Trinidad.

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.

Seroepidemiology of leptospirosis in dogs and rats in Trinidad.

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.

Study on the efficacy of Leptospira vaccines developed from serovars isolated from Trinidad and comparison with commercial vaccines using a hamster model.

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.

Serovars of Leptospira isolated from dogs and rodents.

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.

Analysis of Fc gamma receptor II (CD32) polymorphism in populations of African and South Asian ancestry reveals east-west geographic gradients of allele frequencies.

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.

Natural killer cell immunoglobulin-like receptor (KIR) locus profiles in African and South Asian populations.

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.

Allele frequencies for candidate genes in atherosclerosis and diabetes among Trinidadian neonates.

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.

Polymorphisms in PNLIP, encoding pancreatic lipase, and associations with metabolic traits.

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.

Probing the conformation of the human T-lymphotropic virus I envelope protein complex with monoclonal antibodies.

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.

A truncated HTLV-I envelope protein, lacking the hydrophobic membrane anchor domain, is associated with cellular membranes and virions.

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.

HTLV-1 envelope sequences from Brazil, the Caribbean, and Romania: clustering of sequences according to geographic origin and variability in an antibody epitope.

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.