Phylogeographic reconstruction of the emergence and spread of Powassan virus in the northeastern United States.

Powassan virus is an emerging tick-borne virus of concern for public health, but very little is known about its transmission patterns and ecology. Here, we expanded the genomic dataset by sequencing 279 Powassan viruses isolated from Ixodes scapularis ticks from the northeastern United States. Our phylogeographic reconstructions revealed that Powassan virus lineage II was likely introduced or emerged from a relict population in the Northeast between 1940 and 1975. Sequences strongly clustered by sampling location, suggesting a highly focal geographical distribution. Our analyses further indicated that Powassan virus lineage II emerged in the northeastern United States mostly following a south-to-north pattern, with a weighted lineage dispersal velocity of ~3 km/y. Since the emergence in the Northeast, we found an overall increase in the effective population size of Powassan virus lineage II, but with growth stagnating during recent years. The cascading effect of population expansion of white-tailed deer and I. scapularis populations likely facilitated the emergence of Powassan virus in the northeastern United States.

Phylodynamics of deer tick virus in North America.

The burden of ticks and the pathogens they carry is increasing worldwide. Powassan virus (POWV; Flaviviridae: Flavivirus), the only known North American tick-borne flavivirus, is of particular concern due to rising cases and the severe morbidity of POWV encephalitis. Here, we use a multifaceted approach to evaluate the emergence of the II POWV lineage, known as deer tick virus (DTV), in parts of North America where human cases occur. We detected DTV-positive ticks from eight of twenty locations in the Northeast USA with an average infection rate of 1.4 per cent. High-depth, whole-genome sequencing of eighty-four POWV and DTV samples allowed us to assess geographic and temporal phylodynamics. We observed both stable infection in the Northeast USA and patterns of geographic dispersal within and between regions. A Bayesian skyline analysis demonstrated DTV population expansion over the last 50 years. This is concordant with the documented expansion of Ixodes scapularis tick populations and suggests an increasing risk of human exposure as the vector spreads. Finally, we isolated sixteen novel viruses in cell culture and demonstrated limited genetic change after passage, a valuable resource for future studies investigating this emerging virus.

Jamestown Canyon Virus in Collected Mosquitoes, Maine, United States, 2017-2019.

Jamestown Canyon virus (JCV) is a mosquito-borne arbovirus that circulates in North America. We detected JCV in 4 pools of mosquitoes collected from midcoastal Maine, USA, during 2017-2019. Phylogenetic analysis of a JCV sequence obtained from Aedes cantator mosquitoes clustered within clade A, which also circulates in Connecticut, USA.

Surge in Anaplasmosis Cases in Maine, USA, 2013-2017.

Incidence of human granulocytic anaplasmosis is rising in Maine, USA. This increase may be explained in part by adoption of tick panels as a frequent diagnostic test in persons with febrile illness and in part by range expansion of Ixodes scapularis ticks and zoonotic amplification of Anaplasma phagocytophilum.

Decoupling of Blacklegged Tick Abundance and Lyme Disease Incidence in Southern Maine, USA.

Lyme disease is caused by the bacterial spirochete Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, and Brenner (Spirocheatales: Spirochaetaceae) which is transmitted through the bite of an infected blacklegged tick Ixodes scapularis Say (Ixodida: Ixodidae). Maine, USA, is a high Lyme disease incidence state, with rising incidence of Lyme disease and other tick-borne illnesses associated with increasing I. scapularis abundance and northward range expansion. Members of the public submitted ticks to a tick identification program (1990-2013). From these passive surveillance data, we characterized temporal trends in I. scapularis submission rate (an index of abundance), comparing Maine's northern tier (seven counties) versus southern tier (nine counties). In the northern tier, the I. scapularis submission rate increased throughout the duration of the time series, suggesting I. scapularis was emergent but not established. By contrast, in the southern tier, submission rate increased initially but leveled off after 10-14 yr, suggesting I. scapularis was established by the mid-2000s. Active (field) surveillance data from a site in the southern tier-bird tick burdens and questing adult tick collections-corroborated this leveling pattern. Lyme disease incidence and I. scapularis submission rate were temporally correlated in the northern but not southern tier. This suggested a decoupling of reported disease incidence and entomological risk.

Prevalence and Genetic Characterization of Deer Tick Virus (Powassan Virus, Lineage II) in Ixodes scapularis Ticks Collected in Maine.

Deer tick virus (DTV) is a genetic variant of Powassan virus (POWV) that circulates in North America in an enzootic cycle involving the blacklegged or "deer tick," Ixodes scapularis, and small rodents such as the white-footed mouse. The number of reported human cases with neuroinvasive disease has increased substantially over the past few years, indicating that POWV may be of increasing public health importance. To this end, we sought to estimate POWV infection rates in questing I. scapularis collected from four health districts in Maine (York, Cumberland, Midcoast, and Central Maine). Infection rates were 1.6%, 1.7%, 0.7%, and 0%, respectively, for adults collected from April to November in 2016. Adults collected in October and November in 2017 from York and Cumberland counties had slightly higher rates of 2.3% and 3.5%, respectively. There was no difference in the number of males verses the number of females infected. All positive samples were of the DTV (lineage II) variant. Phylogenetic analysis was performed on 8 of the 15 DTV sequences obtained in 2016. Deer tick virus from the coastal regions were genetically similar and clustered with virus strains isolated from I. scapularis from New York State and Bridgeport, CT. The two inland viruses were genetically nearly identical and grouped with viruses from Massachusetts, Connecticut, and New York. These results are the first reported infection rates and sequences for POWV in questing ticks collected in Maine and will provide a reference point for future POWV studies.

Sugar feeding improves survival of nondiapausing cold-stored Culex pipiens.

The continuous culture of mosquitoes is a costly endeavor for vector biology laboratories. In addition to the resources that must be committed to colony maintenance, biological costs, including genetic drift and accidental colony loss, also can occur. Although alternatives do exist, their application to mosquitoes is limited. Mosquito cryopreservation remains elusive, and many important species lack a well-defined diapause. Previously, we demonstrated that cold storing nondiapausing mated adult females of the northern house mosquito, Culex pipiens L. resulted in a nearly four-fold increase in longevity when measured at the LT50, allowing for cold storage for up to 10 wk. In the current study, we used sugar feeding during cold storage to significantly improve cold storage longevity. At 6 degrees C, the LT50 of cold stored females was 23 wk, and 100% mortality was not realized until 43 wk. Cold-stored females did exhibit reduced fecundity, but egg production returned to normal levels within two generations. These results suggest that cold storage without diapause induction is a viable option for Cx. pipiens, and with the addition of sugar feeding, a colony could be maintained with less than two generations per year.

Cold storage of Culex pipiens in the absence of diapause.

A major expenditure in vector biology laboratories is the rearing of mosquitoes. Most mosquito colonies require substantial effort to maintain, including frequent bloodmeals for optimal performance. Successful cryopreservation of mosquitoes continues to be elusive. Although using diapause as a storage mechanism is an option for mosquito preservation, several obstacles include the lack of a well-characterized diapause or the inability of some species to enter diapause. Thus, other options for preservation are needed. To address this issue, we investigated the use of long-term low-temperature storage in the absence of diapause for adults of the northern house mosquito, Culex pipiens L. Our results indicate that although male longevity is not substantially increased by cold storage, female longevity is dramatically increased by storage at lower temperatures. When mated before storage, females remain reproductively viable after at least 10 wk of storage, although at reduced levels. These results indicate that cold storage without diapause induction is a viable option for colony maintenance in vector biology laboratories.

Isolation of diapause-regulated genes from the flesh fly, Sarcophaga crassipalpis by suppressive subtractive hybridization.

Subtractive suppressive hybridization (SSH) was used to characterize the diapause transcriptome of the flesh fly Sarcophaga crassipalpis. Through these efforts, we isolated 97 unique clones which were used as probes in northern hybridization to assess their expression during diapause. Of these, 17 were confirmed to be diapause upregulated and 1 was diapause downregulated, while 12 were shown to be unaffected by diapause in this species. The diapause upregulated genes fall into several broad categories including heat shock proteins, heavy metal responsive genes, neuropeptides, structural genes, regulatory elements, and several genes of unknown function. In combination with other large-scale analyses of gene expression during diapause, this study assists in the characterization of the S. crassipalpis diapause transcriptome, and begins to identify common elements involved in diapause across diverse taxa.

Up-regulation of heat shock proteins is essential for cold survival during insect diapause.

Diapause, the dormancy common to overwintering insects, evokes a unique pattern of gene expression. In the flesh fly, most, but not all, of the fly's heat shock proteins (Hsps) are up-regulated. The diapause up-regulated Hsps include two members of the Hsp70 family, one member of the Hsp60 family (TCP-1), at least four members of the small Hsp family, and a small Hsp pseudogene. Expression of an Hsp70 cognate, Hsc70, is uninfluenced by diapause, and Hsp90 is actually down-regulated during diapause, thus diapause differs from common stress responses that elicit synchronous up-regulation of all Hsps. Up-regulation of the Hsps begins at the onset of diapause, persists throughout the overwintering period, and ceases within hours after the fly receives the signal to reinitiate development. The up-regulation of Hsps appears to be common to diapause in species representing diverse insect orders including Diptera, Lepidoptera, Coleoptera, and Hymenoptera as well as in diapauses that occur in different developmental stages (embryo, larva, pupa, adult). Suppressing expression of Hsp23 and Hsp70 in flies by using RNAi did not alter the decision to enter diapause or the duration of diapause, but it had a profound effect on the pupa's ability to survive low temperatures. We thus propose that up-regulation of Hsps during diapause is a major factor contributing to cold-hardiness of overwintering insects.

Endemic eastern equine encephalitis in the Amazon region of Peru.

Eastern equine encephalitis virus (EEEV) causes severe neurologic disease in North America, but only two fatal human cases have been documented in South America. To test the hypothesis that alphavirus heterologous antibodies cross-protect, animals were vaccinated against other alphaviruses and challenged up to 3 months later with EEEV. Short-lived cross-protection was detected, even in the absence of cross-neutralizing antibodies. To assess exposure to EEEV in Peru, sera from acutely ill and healthy persons were tested for EEEV and other alphavirus antibodies, as well as for virus isolation. No EEEV was isolated from patients living in an EEEV-enzootic area, and only 2% of individuals with febrile illness had EEEV-reactive IgM. Only 3% of healthy persons from the enzootic region had EEEV-neutralizing antibodies. Our results suggest that humans are exposed but do not develop apparent infection with EEEV because of poor infectivity and/or avirulence of South American strains.

Diapause-specific gene expression in the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization.

In this study we probe the molecular events underpinning diapause observed in overwintering females of Culex pipiens. Using suppressive subtractive hybridization (SSH) we have identified 40 genes that are either upregulated or downregulated during this seasonal period of dormancy. Northern blot hybridizations have confirmed the expression of 32 of our SSH clones, including six genes that are upregulated specifically in early diapause, 17 that are upregulated in late diapause, and two upregulated throughout diapause. In addition, two genes are diapause downregulated and five remain unchanged during diapause. These genes can be categorized into eight functional groups: genes with regulatory functions, metabolically-related genes, those involved in food utilization, stress response genes, cytoskeletal genes, ribosomal genes, transposable elements, and genes with unknown functions.

Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito, Culex pipiens.

Two actin genes cloned from Culex pipiens L. are upregulated during adult diapause. Though actins 1 and 2 were expressed throughout diapause, both genes were most highly expressed early in diapause. These changes in gene expression were accompanied by a conspicuous redistribution of polymerized actin that was most pronounced in the midguts of diapausing mosquitoes that were exposed to low temperature. In nondiapausing mosquitoes reared at 25 degrees C and in diapausing mosquitoes reared at 18 degrees C, polymerized actin was clustered at high concentrations at the intersections of the muscle fibers that form the midgut musculature. When adults 7-10 days post-eclosion were exposed to low temperature (-5 degrees C for 12 h), the polymerized actin was evenly distributed along the muscle fibers in both nondiapausing and diapausing mosquitoes. Exposure of older adults (1 month post-eclosion) to low temperature (-5 degrees C for 12 h) elicited an even greater distribution of polymerized actin, an effect that was especially pronounced in diapausing mosquitoes. These changes in gene expression and actin distribution suggest a role for actins in enhancing survival of diapausing adults during the low temperatures of winter by fortification of the cytoskeleton.

Enhanced cold and desiccation tolerance in diapausing adults of Culex pipiens, and a role for Hsp70 in response to cold shock but not as a component of the diapause program.

Culex pipiens L. reared under diapause-inducing conditions (short daylength; 18 degrees C) were more cold tolerant and desiccation resistant than their nondiapausing counterparts (long daylength; 18 degrees C). Upon cold exposure (-5 degrees C), diapausing mosquitoes reared at 18 degrees C survived nearly twice as long as nondiapausing mosquitoes reared at 18 degrees C and 10 times longer than nondiapausing mosquitoes reared at 25 degrees C. Thus, rearing temperature provided partial protection against low temperature injury in nondiapausing mosquitoes, but maximum resistance to cold was attained by the diapause state. In this species, the supercooling point is not a good indicator of cold tolerance. Both diapausing and nondiapausing females had supercooling points of approximately -16 degrees C, but diapausing as well as nondiapausing females died at temperatures well above the supercooling point, suggesting that low temperature mortality was due to indirect chilling injury. Diapause also conferred greater resistance to desiccation (1.6-2-fold increase in survival) compared with the nondiapause state. The gene encoding a 70-kDa heat shock protein, hsp70, was not up-regulated (i.e., more highly expressed) as a part of the diapause program, nor was it up-regulated by desiccation stress, but it was up-regulated during recovery from cold shock. Cx. pipiens thus differs from a number of other diapausing insect species that are known to developmentally up-regulate hsp70 during diapause.

Diapause in the mosquito Culex pipiens evokes a metabolic switch from blood feeding to sugar gluttony.

A key characteristic of overwintering dormancy (diapause) in the mosquito Culex pipiens is the switch in females from blood feeding to sugar gluttony. We present evidence demonstrating that genes encoding enzymes needed to digest a blood meal (trypsin and a chymotrypsin-like protease) are down-regulated in diapause-destined females, and that concurrently, a gene associated with the accumulation of lipid reserves (fatty acid synthase) is highly up-regulated. As the females then enter diapause, fatty acid synthase is only sporadically expressed, and expression of trypsin and chymotrypsin-like remains undetectable. Late in diapause (2-3 months at 18 degrees C), the genes encoding the digestive enzymes begin to be expressed as the female prepares to take a blood meal upon the termination of diapause. Our results thus underscore a molecular switch that either capacitates the mosquito for blood feeding (nondiapause) or channels the adult mosquito exclusively toward sugar feeding and lipid sequestration (diapause).

Meconial peritrophic matrix structure, formation, and meconial degeneration in mosquito pupae/pharate adults: histological and ultrastructural aspects.

The noncellular peritrophic matrix (PM) that forms around the food bolus in the midgut of many arthropod species may influence the fate of ingested microbes. In mosquitoes, PMs have been identified in the pupal as well as larval and adult stages. In pupae, the PMs surround the meconium, the sloughed larval midgut epithelium. Meconial PM1 (MPM1) forms early in the pupal stadium, and a second meconial PM (MPM2) sometimes forms around the time of adult emergence. A recent study suggests that MPMs contribute to the sterilization of the adult midgut by sequestering microorganisms ingested during the larval stage, which, along with remaining meconial material, are egested after adult emergence. We have compared MPM1 formation and patterns of meconial degeneration in representative species in five mosquito genera and identified a temporal association between MPM1 formation, meconial degeneration, and apolysis. Ultrastructural study of MPM1 and MPM2 in Aedes aegypti (L.) revealed that MPM1 seems to be structurally different from either the larval or adult PMs, whereas MPM2 more closely resembles PM formed around a bloodmeal in adult females. Our results are consistent with the microbial sequestration role.