Serological evidence of widespread exposure of Grenada fruit bats to chikungunya virus.

Antibody detection against selected potentially zoonotic vector-borne alphaviruses and flaviviruses was conducted on sera from bats from all six parishes in Grenada, West Indies. Sera were tested for (i) antibodies to flaviviruses West Nile virus, St. Louis encephalitis virus, Ilhéus virus, Bussuquara virus (BSQV), Rio Bravo virus and all four serotypes of dengue virus (DENV) by plaque reduction neutralization test (PRNT); (ii) antibodies to alphaviruses western equine encephalitis virus, Venezuelan equine encephalitis virus and eastern equine encephalitis virus by epitope-blocking enzyme-linked immunosorbent assay (ELISA); and (iii) antibodies to the alphavirus chikungunya (CHIKV) by PRNT. Two species of fruit bats were sampled, Artibeus jamaicensis and Artibeus lituratus, all roosting in or within 1,000 m of human settlements. Fifteen (36%) of the 42 bats tested for neutralizing antibodies to CHIKV were positive. The CHIKV-seropositive bats lived in localities spanning five of the six parishes. All 43 bats tested for epitope-blocking ELISA antibody to the other alphaviruses were negative, except one positive for Venezuelan equine encephalitis virus. All 50 bats tested for neutralizing antibody to flaviviruses were negative, except one that had a BSQV PRNT80 titre of 20. The CHIKV serology results indicate that bats living close to and within human settlements were exposed to CHIKV in multiple locations. Importantly, bats for this study were trapped a year after the introduction and peak of the human CHIKV epidemic in Grenada. Thus, our data indicate that bats were exposed to CHIKV possibly during a time of marked decline in human cases.

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.

Detection of West Nile virus-specific antibodies and nucleic acid in horses and mosquitoes, respectively, in Nuevo Leon State, northern Mexico, 2006-2007.

In the last 5 years, there has been only one reported human case of West Nile virus (WNV) disease in northern Mexico. To determine if the virus was still circulating in this region, equine and entomological surveillance for WNV was conducted in the state of Nuevo Leon in northern Mexico in 2006 and 2007. A total of 203 horses were serologically assayed for antibodies to WNV using an epitope-blocking enzyme-linked immunosorbent assay (bELISA). Seroprevalences for WNV in horses sampled in 2006 and 2007 were 26% and 45%, respectively. Mosquito collections in 2007 produced 7365 specimens representing 15 species. Culex mosquitoes were screened for WNV RNA and other genera (Mansonia, Anopheles, Aedes, Psorophora and Uranotaenia) were screened for flaviviruses using reverse-transcription (RT)-PCR. Two pools consisting of Culex spp. mosquitoes contained WNV RNA. Molecular species identification revealed that neither pool included Culex quinquefasciatus (Say) (Diptera:Culicidae) complex mosquitoes. No evidence of flaviviruses was found in the other mosquito genera examined. These data provide evidence that WNV is currently circulating in northern Mexico and that non-Cx. quinquefasciatus spp. mosquitoes may be participating in the WNV transmission cycle in this region.

Evaluation of an epitope-blocking enzyme-linked immunosorbent assay for the diagnosis of West Nile virus infections in humans.

An epitope-blocking enzyme-linked immunosorbent assay (b-ELISA) was evaluated for the diagnosis of West Nile virus (WNV) infections in humans. Sera from patients diagnosed with WNV infections from an outbreak in 2003 in Colorado and from patients diagnosed with dengue virus infections from Mexico and Thailand were tested with the b-ELISA. The b-ELISAs were performed using the WNV-specific monoclonal antibody (MAb) 3.1112G and the flavivirus-specific MAb 6B6C-1. Although the WNV-specific b-ELISA was effective in diagnosing WNV infections in humans from Colorado, it was not efficacious for diagnosing WNV infections in serum specimens from Mexico and Thailand. In serum specimens from patients from Colorado, the WNV b-ELISA and the WNV plaque reduction neutralization test showed an overall agreement of 91%. The sensitivity and specificity of the WNV b-ELISA were 89% and 92%, respectively, with a false-positive rate of 5%, based on receiver operating characteristic analysis. In contrast, false-positive rate results in specimens from the countries of Mexico and Thailand, where flaviviruses are endemic, were 79% and 80%, presumably due to the presence of antibodies resulting from previous dengue virus infections in Mexico and/or Japanese encephalitis virus infections or vaccination in Thailand. Thus, in regions where people have experienced previous or multiple flavivirus infections, the use of the b-ELISA for WNV diagnosis is contraindicated.

The Aedes albopictus inhibitor of apoptosis 1 gene protects vertebrate cells from bluetongue virus-induced apoptosis.

We sequenced and characterized the inhibitor of apoptosis (iap) 1 gene from Aedes albopictus, designated as Aaiap1. The Aaiap1 gene rescued Spodoptera frugiperda (Sf9) cells from apoptosis when cotransfected with the Drosophila pro-apoptotic hid gene. The antiapoptotic function of the Aaiap1 gene was evaluated in the bluetongue virus (BTV)-induced apoptosis system. BTV infection induced apoptosis in vertebrate cells via the intrinsic apoptotic pathway. This was shown by the translocation of cytochrome C and the second mitochondria-derived activator of caspase (Smac, also known as DIABLO) from the mitochondria and the subsequent activation of caspase-9 and -3. Stable expression of the Aaiap1 gene in derivative baby hamster kidney cells delayed BTV-induced apoptosis by 24 h and reduced the BTV progeny yield by 10-fold. This study provides the first evidence that the mosquito AaIAP1 protein possesses antiapoptotic activity.

Developmental- and tissue-specific expression of an inhibitor of apoptosis protein 1 homologue from Aedes triseriatus mosquitoes.

We have identified a homologue of the Drosophila inhibitor of apoptosis protein 1 in Aedes triseriatus mosquitoes (designated AtIAP1). The AtIAP1 gene maps to a single locus on chromosome 2. The translation product is a 403 amino acid protein that contains two baculovirus IAP repeat (BIR) domains and a RING finger motif. AtIAP1 mRNA was detectable by RT-PCR amplification in all the mosquito developmental stages (embryos, first-fourth instar larvae, early and late pupae, adults) and adult tissues (midguts, ovaries) examined. In contrast, immunoblots with AtIAP1-specific antibodies revealed that the protein was detectable only in certain developmental stages (first instar larvae, early pupae, adults) and tissues (ovaries). AtIAP1-specific serum also recognized proteins in Ae. aegypti, Ae. albopictus and Culex tritaeniorhynchus. Immunoblot analysis revealed that similar amounts of IAP1 were expressed in LaCrosse virus infected and uninfected Ae. albopictus cell cultures.

Identification and sequence determination of mRNAs detected in dormant (diapausing) Aedes triseriatus mosquito embryos.

Many insects survive adverse climatic conditions in a dormant state known as diapause. In this study, we identified and sequenced several mRNAs in diapausing Aedes triseriatus mosquito embryos. Using reverse-transcription PCR and 5' RACE, we identified a 995-nucleotide cDNA that encodes a 259-amino acid protein of unknown function. This putative protein displays strong sequence similarity to Drosophila melanogaster (95%), human (87%), Caenorhabditis elegans (86%) and yeast (81%) counterparts. The second identified full-length cDNA consists of 624 nucleotides and encodes a 174-amino acid protein of unknown function. This putative protein displays significant sequence similarity to D. melanogaster (68%), human (59%), plant (57%) and yeast (49%) counterparts. We also detected a number of cDNA fragments that exhibited significant sequence similarity to a mitochondrial cytochrome C oxidase subunit, human N33 protein (a potential human prostate tumor suppressor), 18S and 28S ribosomal RNAs, protein disulfide-isomerase, and guanine nucleotide-binding protein.

Complete cDNA and deduced amino acid sequence of the chaperonin containing T-complex polypeptide 1 (CCT) delta subunit from Aedes triseriatus mosquitoes.

The chaperonin containing t-complex polypeptide 1 (CCT) assists in the ATP-dependent folding and assembly of newly translated actin and tubulin in the eukaryotic cytosol. CCT is composed of eight different subunits, each encoded by an independent gene. In this report, we used RT-PCR amplification and 5'- and 3'-rapid amplification of cDNA ends (RACE) to determine the complete cDNA sequence of the CCT delta subunit from Aedes triseriatus mosquitoes. The CCT delta cDNA is 1936 nucleotides in length and encodes a putative 533 amino acid protein with a calculated molecular mass of 57,179 daltons and pI of 7.15. Hydrophobic residues comprise 39.8% of the amino acid sequence and putative motifs for ATP-binding and ATPase-activity are present. The amino acid sequence displays strong sequence similarity to Drosophila melanogaster (92%), human (85%), puffer fish (84%) and mouse (84%) counterparts. CCT delta mRNA was detected in both biosynthetically active (embryonating) and dormant (diapausing) Ae. triseriatus embryos by RT-PCR analysis.

Molecular cloning and complete cDNA sequences of the ribosomal proteins rpl34 and rpl44 from Aedes triseriatus mosquitoes.

We present the complete cDNA and deduced amino acid sequences of the 60S ribosomal subunit proteins, rpL34 and rpL44, from Aedes triseriatus mosquitoes. The rpL34 cDNA is 554 nucleotides in length and encodes a 139 amino acid protein with a calculated molecular mass of 15 732 daltons. The putative protein displays strong sequence similarity to rpL34 of Aedes albopictus mosquitoes (92%), humans (60%) and rats (58%). The protein is highly basic and contains a C-terminal repetitive-alanine domain and four putative nucleolar localization signals. The rpL44 cDNA consists of 450 nucleotides and encodes a 104 amino acid protein with a calculated molecular mass of 12 544 daltons. The putative protein displays strong sequence similarity to rpL44 of Brugia malayi (87%), Caenorhabditis elegans (86%) and humans (85%). The protein is highly basic and contains a putative nucleolar localization signal. The mRNAs for both rpL34 and rpL44 were detected in biosynthetically active (embryonating) and dormant (diapausing) Ae. triseriatus embryos by RT-PCR analysis.

Identification and analysis of truncated and elongated species of the flavivirus NS1 protein.

The flavivirus non-structural glycoprotein NS1 is often detected in Western blots as a heterogeneous cluster of bands due to glycosylation variations, precursor-product relationships and/or alternative cleavage sites in the viral polyprotein. In this study, we determined the basis of structural heterogeneity of the NS1 protein of Murray Valley encephalitis virus (MVE) by glycosylation analysis, pulse-chase experiments and terminal amino acid sequencing. Inhibition of N-linked glycosylation by tunicamycin revealed that NS1 synthesised in MVE-infected C6/36 cells was derived from two polypeptide backbones of 39 kDa (NS1(o)) and 47 kDa (NS1'). Pulse-chase experiments established that no precursor-product relationship existed between NS1(o) and NS1' and that both were stable end products. Terminal sequencing revealed that the N- and C-termini of NS1(o) were located at amino acid positions 714 and 1145 in the polyprotein respectively, consistent with the predicted sites based upon sequence homology with other flaviviruses. Expression of the NS1 gene alone or in conjunction with NS2A by recombinant baculoviruses demonstrated that the production of NS1' was dependent on the presence of NS2A, indicating that the C-terminus of the larger protein was generated within NS2A. A smaller form (31 kDa) of NS1 (deltaNS1) was also identified in MVE-infected Vero cultures, and amino acid sequencing revealed a 120-residue truncation at the N-terminus of this protein. This corresponds closely with the in-frame 121-codon deletion at the 5' end of the NS1 gene of defective MVE viral RNA (described by Lancaster et al. in 1998), suggesting that deltaNS1 may be a translation product of defective viral RNA.

A novel complex formed between the flavivirus E and NS1 proteins: analysis of its structure and function.

We examined the structural features and functional significance of a novel complex which forms between the envelope (E) protein and nonstructural protein NS1 of Murray Valley encephalitis (MVE) virus. Western blot analysis of virus-infected C6/36 cell lysates revealed that the undenatured form of this E-NS1 complex was a heat-sensitive E-(NS1 dimer) complex. Furthermore, the E-NS1 complex was observed in cells infected with Kunjin, Japanese encephalitis, West-Nile and Kokobera viruses which indicates the complex is a common feature of flavivirus infection. E-NS1 complex which had been immunoaffinity purified from MVE-infected cell lysates or eluted from gel slices exhibited partial breakdown into the individual monomers, demonstrating that the complex arose from the association of E and NS1 proteins and was not a single polypeptide created from incomplete gene cleavage. Radioimmunoprecipitation and western blot analysis of MVE-infected cell lysates and culture fluid preparations collected at various times after infection revealed that the E-NS1 complex has a long half life, accumulates in the virus-infected cell with time and is not secreted into the extracellular fluid. We have postulated that the E-NS1 complex, or at least a major portion of the complex, is a non-specific aggregation with no functional significance in the viral life cycle.