Characterization of B-cell Responses to Zika Virus (ZIKV)

Objective: Most ZIKV infections occur in regions endemic for the related dengue virus (DENV). Anti-DENV antibodies have been demonstrated to cross-react with ZIKV. Some neutralize ZIKV infection while others mediate antibody-dependent enhancement (ADE), exacerbating ZIKV infection and complicating diagnosis of the etiologic agent. We aimed to characterize the humoral immune response in a ZIKV+, DENV- experienced individual in order to explore this anamnestic response and identify antibodies that may be useful in the development of therapeutic agents. Design and Methodology: Peripheral blood mononuclear cells (PBMCs) were collected from an individual (TT66) who was newly infected with ZIKV but had two previous DENV infections. Plasmablasts were isolated and analyses conducted using Atreca's Immune Repertoire CaptureTM technology. Monoclonal antibodies (mAbs) derived from TT66 during their acute and convalescent phase of ZIKV infection were screened in vitro for ZIKV and DENV binding and neutralization activity. Epitopes were then mapped using a shotgun mutagenesis approach. Results: We observed clonal expansion of two distinct antibody lineages representing 70% of total immunoglobulin sequences from TT66. We screened 18 mAbs representing two major lineages and five smaller families for neutralization and ADE between DENV and ZIKV. No highly typespecific mAbs were observed but rather a diverse pattern of neutralization, even within an individual lineage. Shotgun mutagenesis epitope mapping demonstrated epitopes for two of these broadly neutralizing mAb lineages lay within domain II ofE, close to the fusion loop. Conclusions: Results suggest that neutralizing antibody responses to ZIKV are extensively shaped by previous DENV exposure.

Anti-Budding Assay: Development of a high throughput screen to identify neutralizing antibodies that block Chikungunya virus (CHIKV) release

Objective: No licensed CHIKV vaccines or effective therapeutic agents are currently available. However, some CHIKV-specific monoclonal antibodies (mAbs) are highly effective in animal models, both prophylactically and therapeutically. This is thought to be largely mediated via blocking of CHIKV entry into cells. However, we and others have shown that inhibition of viral release/ budding is also a major mechanism for CHIKV control. We aimed to develop a high throughput in vitro screening assay to efficiently identify "antibudding"mAbs. Design and Methodology: An assay for quantification of viral budding from infected cells was optimised by varying cell line, cell density, multiplicity of infection (MOI), incubation periods, NH4Cl concentration and plate type. The assay utilized our novel, fully replication -competent, attenuated CHIKV nano-luciferase (nluc) reporter virus (CHIKV 181/25 E2nluc). The optimised assay was used to screen CHIKV+, Zika virus (ZIKV)+, CHIKV-/ZIKV- sera, and cloned memory B-cells from a CHIKV+ individual. Results: Optimal conditions involved use of rhabdomyosarcoma (RD) cells, bulk-infected at MOI 1 for 2hrs, removal of residual virus, resuspension in media containing 20mM NH4Cl, seeding at 2.5x104cells/well into 96-well plates and luminometry after 18hrs. Inter-plate coefficient of variability CV scores and Z' values remained <15% and >0.5 respectively, indicative of a valid assay. Most CHIKV+ sera displayed potent antibudding activity, two displayed no significant activity, and there was no ZIKV cross-reactivity. Of 800 memory B-cell clones, 13 exhibited significant anti-budding antibody activity. Conclusions: We developed a sensitive, reproducible, Biosafety level (BSL-2) safe, high throughput CHIKV antibudding assay useful for screening both polyclonal sera and monoclonal antibodies.

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.

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.

A retrospective study of the 1996 DEN-1 epidemic in Trinidad: demographic and clinical features

A retrospective analysis of the 1996 DEN-1 epidemic in Trinidad was undertaken to better understand the clinical and demographic expression of dengue infection in the island during one of the larger epidemics in the past 10 years and following the reintroduction of DEN-1 into the island in 1991 after a gap of 14 years. A total of 393 laboratory-confirmed cases were identified. Of these, notes for 157 patients were available for analysis. The epidemic was island-wide, though most cases occurred in the most densely populated county of St. George. There was a slight predominance of females (51.6 per cent) among the cases, and while all age groups were affected, older children and adults comprised the majority. South Asians among the population predominated. Overall, 27 clinical symptoms were reported. The most common were: fever (98.7 per cent), generalized pain (96.2 per cent) and anorexia (63.1 per cent). Rash, arthralgia, retro-orbital pain and haemorrhage (all mentioned in the WHO clinical description for dengue fever) were reported in <50 per cent of cases. Gastrointestinal symptoms were also very common and occurred in over two-thirds of cases at presentation. Bleeding manifestations were reported in 30 per cent of patients and commonly involved the gastrointestinal tract. Features of DHF were noted in only six (4 per cent)

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.

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.

Nitric oxide levels in children with febrile illnesses - Poster abstract

OBJECTIVE: To determine nitric oxide (NO) levels in children with febrile illness and to investigate its prognostic value in early dengue virus infections. DESIGN AND METHOD: A pilot case control study was conducted from September 1998 to March 1999 at the Eric Williams Medical Sciences Complex, Priority Care Facility (PCF), in Trinidad. Serum NO concentrations were measured in children <12 years presenting with febrile illnesses in the absence of clinically significant bacterial infection. RESULTS: Of 59 blood samples collected, there were 22 from individuals diagnoses as virally induced gastroenteritis (VGE), with 31 non-specific viral illnesses (VI), and 6 viral infections associated with upper-respiratory tract infection (URTI-VI). The mean (NO) in all cases was above normal (11.9 uM SD 5.9) 81.17 uM in VGE, 41.30 uM in VI and 57.83 uM in URTI-VI. Krushkal-Wallis test revealed that there was a significant difference (p = 0.007) between all three means and that the mean (NO) for VGE was significantly higher (p = 0.003) than that of VI. CONCLUSIONS: The degree of elevation of NO varies between viral illnesses in children and its potential as a rapid prognostic marker warrants further investigation.(AU)

A preliminary study of neopterin as a potential marker for severe dengue virus infection

Dengue virus cause the non-fatal dengue fever and the life-threating dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). Early clinical features of DF and DHF/DSS are indistinguishable and it is difficult to identify early DHF/DSS using clinical and laboratory parameters. A prospective observational study (KALAYANAROOJ et al, 1997) of Thai children with fever for less than 72h revealed that, at enrolment, plasma aspartate aminotransferase (AST)levels were significantly raised in children who developed DHF but were of relatively low positive predictive value (PPV=0.27 for AST>40U/mL). MONATH (1997), in his review of the paper by Kalayanaroo et al., noted the lack of 'clear clinical prognostic indicators for DHF' and called for strategies to improve diagnosis (AU)