Dongyoung Shin

Genome Sequence Analysis of Dengue Virus 1 Isolated in Key West, Florida

Dengue virus (DENV) is transmitted to humans through the bite of mosquitoes. In November 2010, a dengue outbreak was reported in Monroe County in southern Florida (FL), including greater than 20 confirmed human cases. The virus collected from the human cases was verified as DENV serotype 1 (DENV-1) and one isolate was provided for sequence analysis. RNA was extracted from the DENV-1 isolate and was used in reverse transcription polymerase chain reaction (RT-PCR) to amplify PCR fragments to sequence. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the entire genome. The DENV-1 isolate found in Key West (KW), FL was sequenced for whole genome characterization. Sequence assembly, Genbank searches, and recombination analyses were performed to verify the identity of the genome sequences and to determine percent similarity to known DENV-1 sequences. We show that the KW DENV-1 strain is 99% identical to Nicaraguan and Mexican DENV-1 strains. Phylogenetic and recombination analyses suggest that the DENV-1 isolated in KW originated from Nicaragua (NI) and the KW strain may circulate in KW. Also, recombination analysis results detected recombination events in the KW strain compared to DENV-1 strains from Puerto Rico. We evaluate the relative growth of KW strain of DENV-1 compared to other dengue viruses to determine whether the underlying genetics of the strain is associated with a replicative advantage, an important consideration since local transmission of DENV may result because domestic tourism can spread DENVs.

Transcriptomics of differential vector competence: West Nile virus infection in two populations of Culex pipiens quinquefasciatus linked to ovary development

BackgroundUnderstanding mechanisms that contribute to viral dissemination in mosquito vectors will contribute to our ability to interfere with the transmission of viral pathogens that impact public health. The expression of genes in two Culex pipiens quinquefasciatus populations from Florida with known differences in vector competence to West Nile virus (WNV) were compared using high throughput sequencing.ResultsA total of 15,176 transcripts were combined for comparison of expression differences between the two populations and 118 transcripts were differentially expressed (p < 0.05). The fold change in expression of the differentially expressed genes ranged from -7.5 – 6.13. The more competent population for WNV (Gainesville) over expressed 77 genes and down regulated 44 genes, compared with the less competent population for WNV (Vero Beach). Also, splicing analysis identified 3 transcripts with significantly different splice forms between the two populations. The functional analysis showed that the largest proportion of transcripts was included in the catalytic activity and transporter activity groups except for those in the unknown group. Interestingly, the up- regulated gene set contained most of the catalytic activity function and the down- regulated gene set had a notable proportion of transcripts with transporter activity function. Immune response category was shown in only the down regulated gene set, although those represent a relatively small portion of the function. Several different vitellogenin genes were expressed differentially. Based on the RNAseq data analysis, ovary development was compared across the populations and following WNV infection. There were significant differences among the compared groups.ConclusionsThis study suggests that ovary development is correlated to vector competence in two Culex populations in Florida. Both populations control energy allocations to reproduction as a response to WNV. This result provides novel insight into the defense mechanism used by Culex spp. mosquitoes against WNV.

Evidence of Zika Virus RNA Fragments in Aedes albopictus (Diptera: Culicidae) Field-Collected Eggs From Camaçari, Bahia, Brazil

Abstract A major mosquito-borne viral disease outbreak caused by Zika virus (ZIKV) occurred in Bahia, Brazil, in 2015, largely due to transmission by the mosquito, Aedes aegypti (L.). Detecting ZIKV in field samples of Ae. aegypti has proven problematic in some locations, suggesting other mosquito species might be contributing to the spread of ZIKV. In this study, several (five) adult Aedes albopictus (Skuse) mosquitoes that emerged from a 2015 field collection of eggs from Camaçari, Bahia, Brazil, were positive for ZIKV RNA; however, attempts to isolate live virus were not successful. Results from this study suggest that field-collected Ae. albopictus eggs may contain ZIKV RNA that require further tests for infectious ZIKV. There is a need to investigate the role of Ae. albopictus in the ZIKV infection process in Brazil and to study the potential presence of vertical and sexual transmission of ZIKV in this species.

Susceptibility of Florida Aedes aegypti and Aedes albopictus to dengue viruses from Puerto Rico

ABSTRACT: Locally acquired dengue cases in the continental U.S. are rare. However, outbreaks of dengue-1 during 2009, 2010, and 2013 in Florida and dengue-1 and -2 in Texas suggest vulnerability to transmission. Travel and commerce between Puerto Rico and the U.S. mainland is common, which may pose a risk for traveler-imported dengue cases. Mosquitoes were collected in Florida and used to evaluate their susceptibility to dengue viruses (DENV) from Puerto Rico. Aedes aegypti and Ae. albopictus were susceptible to virus infection with DENV-1 and -2. No significant differences were observed in rates of midgut infection or dissemination between Ae. aegypti or Ae. albopictus for DENV-1 (6–14%). Aedes aegypti was significantly more susceptible to midgut infection with DENV-2 than Ae. albopictus (Ae. aegypti, ∼28%; Ae. albopictus, ∼9%). The dissemination rate with dengue-2 virus for Ae. aegypti (23%) was greater than Ae. albopictus (0%), suggesting that Ae. albopictus is not likely to be an important transmitter of the DENV-2 isolate from Puerto Rico. These results are discussed in light of Floridas vulnerability to DENV transmission.

Elucidation of the genetic differences in Trialeurodes vaporariorum populations under vegetable greenhouse conditions by using the allozyme approach

Genetic differentiation of Trialeurodes vaporariorum (Westwood 1856) populations was examined using biochemical and allozyme analysis. For biochemical analysis, general esterase and glutathion‐S‐transferase were tested. Allozyme genetic variability in 11 populations of T. vaporariorum was investigated using five loci from four enzyme systems. Although there are large variations between populations, T. vaporariorum is grouped into two populations with geographic barriers, based on Neis genetic distance in the Baekdudaegan Mountains. Within these two groups, low migration and linkage disequilibrium reveal that populations tend to be influenced by gene drift rather than uniform selection pressures. The effect of genetic drift is greater than the effect of uniform selection by insecticides or host plant resistance, which is suggested by the FST estimates in this study. Based on this basic research, more effective whitefly control programs could be built in the future.

Culex interrogator (Diptera: Culicidae), a Mosquito Species New to Florida

In recent years, two mosquito species, Culex declarator Dyar and Knab and Culex coronator Dyar and Knab, have been detected for the first time in Florida. Single specimens of Cx declarator were collected in 1998 and 2002 in Indian River County and many more Cx. declarator adults were encountered in county surveillance activities during 2003 (Darsie and Shroyer 2004). However, since 2003 this mosquito has been collected infrequently and has been reported from only one additional Florida county, Monroe. Culex coronator is thought to have spread eastward from Texas (Gray et al. 2008) and was initially found in the western panhandle section of Florida in 2005 (Smith et al. 2006). Unlike Cx. declarator, Cx coronator is now found in nearly all Florida counties and is especially common in the southern part of the state (Connelly et al. 2016). Here we report on the finding of yet another Culex spp., Culex interrogator (Dyar and Knab), which is new to Florida. Historically, the northern geographic distribution limit of all three of these Culex (Culex) mosquitoes was southern Texas and other states bordering Mexico (Carpenter and LaCasse 1955, Darsie and Ward 1981). Culex interrogator was initially discovered in Florida in Broward County (Lat. 26°01’10.0”N, Long. 80°07’16.1”W) in August, 2013 when several larvae of this species were collected from a storm water drain that was 53 cm in diameter and contained a considerable amount of grass cuttings. Cooccurring with Cx. interrogator were three other mosquito species: Culex nigripalpus (Theobald), Culex quinquefasciatus (Say), and Culex salinarius (Coquillett). A second Broward County site with Cx. interrogator was found in October, 2014, when immatures of this species were collected from a roadside swale. This site was approximately 26 km west of the first site. In July and August, 2014 at two field study sites in Indian River County, more than 3,000 Culex egg rafts were collected from two types of ovitraps (busboy trays, 56 cm x 44 cm, 8 cm depth) that had been deployed to assess oviposition site preferences of Culex spp. Egg rafts were returned to Florida Medical Entomology Laboratory (FMEL) and held in insectary conditions at 28° C and 80% humidity. After eggs hatched, larvae were identified to species based on larval morphological features. Nine of these egg rafts, all from a single study site, produced Cx. interrogator. Other mosquito species collected with Cx. interrogator in the ovitraps included Cx. nigripalpus, Cx. quinquefasciatus, Cx. coronator, and Cx. salinarius. The ovitraps with Cx. interrogator egg rafts were located in a subdivision with numerous undeveloped lots bordering on agricultural lands. During the summer and autumn of 2014, Cx. interrogator adults were collected at seven other locations in Indian River County, including six of the eight sentinel chicken sites operated by the Indian River Mosquito Control District (IRMCD) where mosquitoes were captured in chicken coop exit traps. In the southern part of the county, near the edge of a pasture, several Cx. interrogator were captured in a dry-ice baited CDC miniature light trap and a passive box trap (Ritchie et al. 2013). Many more were taken in early morning collections of resting adults made with a battery powered aspirator (Table 1). More recently, Cx interrogator has been detected in two other Florida counties. In August, 2015 adults of this mosquito were collected in a CO2-baited light trap near Okeechobee City, Okeechobee County. In January, 2016, immature Cx. interrogator were found in a sample taken from a roadside swale with grass cuttings in Citrus County (Figure 1). Culex interrogator larvae are readily distinguished from

Assessment of esterase gene expression as a risk marker for insecticide resistance in Florida Culex nigripalpus (Diptera: Culicidae).

ABSTRACT Esterases are enzymatic proteins known to play a role in insecticide resistance formation. To further our understanding of the development of insecticide resistance, we tested the gene expression level of a gene implicated in insecticide resistance (Temsha est-1) from Culex nigripalpus Theobald (Diptera: Culicidae) in field mosquitoes. We found that the level of expression of TE-1 differed depending on the frequency of exposure to organophosphate insecticide through expression studies. Temsha est-1 cDNA is 1,808 base pairs and fully sequenced with up to 96% nucleotide sequence identity to esterase B genes of other mosquito species. The genes from five different species, including TE-1, were closely related by genetic distance and phylogenetic analysis. Differential expression of this gene that is correlated to differences in susceptibility towards organophosphate would provide the ability to use Temsha est-1 as an indicator of the formation of tolerance/resistance. This would greatly enhance mosquito control efforts by allowing targeted application of insecticides to mosquito populations that are most susceptible. Also, it would provide resistance information so that a rational design could be used for insecticide rotation schedules.

The Effect of West Nile Virus Infection on the Midgut Gene Expression of Culex pipiens quinquefasciatus Say (Diptera: Culicidae)

The interaction of the mosquito and the invading virus is complex and can result in physiological and gene expression alterations in the insect. The association of West Nile virus (WNV) and Culex pipiens quinquefasciatus mosquitoes results in measurable changes in gene expression; 22 gene products were shown previously to have altered expression. Sequence analysis of one product, CQ G1A1, revealed 100% amino acid identity to gram negative bacteria binding proteins (CPQGBP) in Cx. p. quinquefasciatus, Aedes aegypti (70%) and Anopheles gambiae (63%) that function in pathogen recognition. CQ G1A1 also was differentially expressed following WNV infection in two populations of Cx. p. quinquefasciatus colonized from Florida with known differences in vector competence for WNV and showed spatial and temporal gene expression differences in midgut, thorax, and carcass tissues. These data suggest gene expression of CQ G1A1 is influenced by WNV infection and the WNV infection-controlled expression differs between populations and tissues.

The Effect of Permethrin Resistance on Aedes aegypti Transcriptome Following Ingestion of Zika Virus Infected Blood

Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance among mosquito populations is a consequence of the application of insecticides for mosquito control. We used RNA-sequencing to compare transcriptomes between permethrin resistant and susceptible strains of Florida Ae. aegypti in response to Zika virus infection. A total of 2459 transcripts were expressed at significantly different levels between resistant and susceptible Ae. aegypti. Gene ontology analysis placed these genes into seven categories of biological processes. The 863 transcripts were expressed at significantly different levels between the two mosquito strains (up/down regulated) more than 2-fold. Quantitative real-time PCR analysis was used to validate the Zika-infection response. Our results suggested a highly overexpressed P450, with AAEL014617 and AAEL006798 as potential candidates for the molecular mechanism of permethrin resistance in Ae. aegypti. Our findings indicated that most detoxification enzymes and immune system enzymes altered their gene expression between the two strains of Ae. aegypti in response to Zika virus infection. Understanding the interactions of arboviruses with resistant mosquito vectors at the molecular level allows for the possible development of new approaches in mitigating arbovirus transmission. This information sheds light on Zika-induced changes in insecticide resistant Ae. aegypti with implications for mosquito control strategies.

Culex quinquefasciatus (Diptera: Culicidae) From Florida Transmitted Zika Virus

We report a laboratory colony of Culex quinquefasciatus mosquitoes were experimentally able to salivate Zika virus (ZIKV, Flaviviridae; Flavivirus) at 16 days post infection (dpi). ZIKV RNA was detected in bodies and in saliva deposited on filter paper cards with subsequent studies demonstrating the presence of live ZIKV in saliva.