No assembly required: Full-length MHC class I allele discovery by PacBio circular consensus sequencing.

Single-molecule real-time (SMRT) sequencing technology with the Pacific Biosciences (PacBio) RS II platform offers the potential to obtain full-length coding regions (∼1100-bp) from MHC class I cDNAs. Despite the relatively high error rate associated with SMRT technology, high quality sequences can be obtained by circular consensus sequencing (CCS) due to the random nature of the error profile. In the present study we first validated the ability of SMRT-CCS to accurately identify class I transcripts in Mauritian-origin cynomolgus macaques (Macaca fascicularis) that have been characterized previously by cloning and Sanger-based sequencing as well as pyrosequencing approaches. We then applied this SMRT-CCS method to characterize 60 novel full-length class I transcript sequences expressed by a cohort of cynomolgus macaques from China. The SMRT-CCS method described here provides a straightforward protocol for characterization of unfragmented single-molecule cDNA transcripts that will potentially revolutionize MHC class I allele discovery in nonhuman primates and other species.

Exposure to chikungunya virus and adult longevity in Aedes aegypti (L.) and Aedes albopictus (Skuse).

Chikungunya virus (CHIKV) recently emerged as a global threat to public health through its adaptation to the cosmopolitan mosquito Aedes albopictus Skuse. Aedes albopictus is highly susceptible to the emergent strain of CHIKV, relative to the historical vector of CHIKV, Aedes aegypti (L.). We hypothesized that the high susceptibility of Ae. albopictus to CHIKV may have a cost in terms of longevity and fecundity among infected vs non-infected mosquitoes, relative to Ae. aegypti. We performed a longevity experiment comparing Ae. aegypti and Ae. albopictus exposed to the emergent strain of CHIKV (LR-2006OPY1). We found a small but significant decrease in longevity of Ae. albopictus, but not Ae. aegypti, in response to exposure to CHIKV. We did not observe significant differences in numbers of eggs laid by either species in response to exposure. Longevity and body titer of infected Ae. albopictus were significantly negatively correlated, such that individuals that lived longer had lower viral body titers when they died. The cost of exposure, while not high, suggests there may be physiological constraints in the evolution of viral infectiousness in its insect vector.

Larval environmental temperature and the susceptibility of Aedes albopictus Skuse (Diptera: Culicidae) to Chikungunya virus.

A key feature in the recent widespread epidemic of the mosquito-borne alphavirus chikungunya virus (CHIKV) was the important role of Aedes albopictus, formerly regarded as a secondary vector, compared to the presumed primary vector Aedes aegypti. Ae. albopictus, a container-inhabiting mosquito, is an invasive species that occurs over a wide geographic range spanning tropical and temperate latitudes. In this study we examine the effects of a broad range of larval rearing temperatures on CHIKV infection, dissemination, and viral titer in Florida F(1) Ae. albopictus. Adults from larvae reared at 18 degrees C, 24 degrees C, and 32 degrees C differed significantly in size, development time, and CHIKV infection rate. Adult females with the largest body size were produced from the coolest temperature, took the longest to mature, and six times more likely to be infected with CHIKV than females reared at 32 degrees C. There was also a significant effect of rearing temperature on viral dissemination, resulting in an increase in population dissemination at the coolest temperature. This study indicates that climate factors, such as temperature, experienced at the larval stage, can influence the competence of adult females to vector arboviruses.

Effects of infectious virus dose and bloodmeal delivery method on susceptibility of Aedes aegypti and Aedes albopictus to chikungunya virus.

Chikungunya virus (CHIKV) is an arbovirus (genus Alphavirus, family Togaviridae) that has recently caused disease outbreaks in the Indian Ocean basin and southern Europe. These outbreaks could be associated with a possible shift in primary vector from Aedes aegypti to Ae. albopictus. To evaluate vector competence differences in possible CHIKV vectors, we evaluated the dose-dependant susceptibility of Florida strains of Ae. albopictus and Ae. aegypti for infection with a La Réunion island strain of CHIKV. Pledget and water-jacketed membrane feeding systems were also evaluated. We show that both Aedes spp. were susceptible to the highest CHIKV doses, whereas only Ae. albopictus developed disseminated infections after exposure to the two lowest doses. Infection rates for both mosquito species were significantly affected by the bloodmeal delivery method used. This information is important in assessing risk of an outbreak of imported CHIKV in the United States, in determining differences in vectorial capacity of these two vector species, and in evaluating arbovirus delivery methods in the laboratory.

Susceptibility of Florida mosquitoes to infection with chikungunya virus.

Chikungunya virus (CHIKV) has caused recent, large epidemics on islands in the Indian Ocean, raising the possibility of more widespread CHIKV epidemics. Historically, CHIKV has been vectored by Aedes aegypti, but these outbreaks likely also involved Ae. albopictus. To examine the potential for an outbreak of CHIKV in Florida, we determined the susceptibility to CHIKV of F1 Ae. aegypti and Ae. albopictus from Florida. In addition, we also evaluated two well-characterized laboratory strains (Rockefeller and Lake Charles) of these species. We determined infection and dissemination rates as well as total body titer of mosquitoes 7 days post-exposure (pe) (Ae. albopictus) and 3, 7, and 10 days pe (Ae. aegypti). All mosquito strains were susceptible to both infection and dissemination, with some variation between strains. Our results suggest Florida would be vulnerable to transmission of CHIKV in urban and rural areas where the two vector species occur.

Evidence for a population expansion in the West Nile virus vector Culex tarsalis.

Population genetic structure of the West Nile Virus vector Culex tarsalis was investigated in 5 states in the western United States using 5 microsatellite loci and a fragment of the mitochondrial reduced form of nicotinamide adenine dinucleotide dehydrogenase 4 (ND4) gene. ND4 sequence analysis revealed a lack of isolation by distance, panmixia across all populations, an excess of rare haplotypes, and a star-like phylogeny. Microsatellites revealed moderate genetic differentiation and isolation by distance, with the largest genetic distance occurring between populations in southern California and New Mexico (F(ST) = 0.146). Clustering analysis and analysis of molecular variance on microsatellite data indicated the presence of 3 broad population clusters. Mismatch distributions and site-frequency spectra derived from mitochondrial ND4 sequences displayed pattern's characteristic of population expansion. Fu and Li's D* and F*, Fu's F(S), and Tajima's D statistics performed on ND4 sequences all revealed significant, negative deviations from mutation-drift equilibrium. Microsatellite-based multilocus heterozygosity tests showed evidence of range expansion in the majority of populations. Our results suggest that C. tarsalis underwent a range expansion across the western United States within the last 375,000-560,000 years, which may have been associated with Pleistocene glaciation events that occurred in the midwestern and western United States between 350,000 and 1 MYA.