Progress 08/01/09 to 09/30/14
Outputs
Target Audience: The target audiences include: medical entomologists, virologists, mosquito control personnel, federal, state and local public health officials. Efforts to deliver science-based knowledge to stakeholders included publication of results in peer-reviewed scientific journals (Journal of Medical Entomology, New England Journal of Medicine, Proceedings Royal Society, London and Vector-Borne and Zoonotic Diseases) that have world-wide distribution to infectious disease specialists, medical entomologists and public health officials. Oral presentations were given at regional, national and international scientific conferences of vector-borne disease mosquito control organizations, and in interviews with local newspaper, radio and television reporters from Connecticut. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Results have been published or are in-press in peer-reviewed scientific journals: Vector-Borne and Zoonotic Diseases and American Journal of Tropical Medicine and Hygiene for dissemination to the scientific community. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1 Issue: Cache Valley (CV) virus is a mosquito-borne bunyavirus (family Bunyaviridae, genus Orthobunyavirus) that is enzootic throughout much of North and Central America. White-tailed deer, Odocoileus virginianus have been incriminated as important reservoir and amplification hosts and the virus has been found in a diverse array of mosquito species but the principal vectors are unknown. Response: A 16-year study was undertaken to identify the primary mosquito vectors in Connecticut, quantify seasonal prevalence rates of infection and define the spatial geographic distribution of CV virus in the state as a function of land use and white-tailed deer populations which have increased substantially over this period. Cache Valley virus was isolated from 16 mosquito species in 7 genera, almost all of which were multivoltine and mammalophilic. Anopheles punctipennis was incriminated as the most consistent and likely vector in this region based on yearly isolation frequencies and the spatial geographic distribution of infected mosquitoes. Other species exhibiting frequent temporal and moderate, spatial geographic patterns of virus isolation within the state included Ochlerotatus trivittatus, Ochlerotatus canadensis, Aedes vexans and Aedes cinereus. New isolation records for CV virus were established for Anopheles walkeri, Culiseta melanura, and Ochlerotatus cantator. Other species from which CV virus was isolated included Anopheles quadrimaculatus, Coquillettidia perturbans, Culex salinarius, Ochlerotatus japonicus, Ochlerotatus sollicitans, Ochlerotatus taeniorhynchus, Ochlerotatus triseriatus and Psorophora ferox. Mosquitoes infected with CV virus were equally distributed throughout urban, suburban and rural locales, and infection rates were not directly associated with the localized abundance of white-tailed deer possibly due to their saturation throughout the region. Virus activity in mosquitoes was episodic with no consistent pattern from year to year and fluctuations in yearly seasonal infection rates did not appear to be directly impacted by overall mosquito abundance. However, the years in which the greatest number of CV virus isolations and highest prevalence rates of infection were observed were associated with excessively wet summers resulting in exceptionally high mosquito populations, thus suggesting a possible link. Virus infection in mosquitoes occurred late in the season that mostly extended from mid-August through September when adult mosquito populations were visibly declining and were comparatively low.Impact:In this investigation, we have shown that CV virus is widely distributed throughout Connecticut, infects a diverse array of mosquitoes but occurs sporadically with no consistent pattern of activity from one year to the next. Anopheles punctipennis was incriminated as themain vector in this region based on yearly isolation frequencies and the spatial geographic distribution of infected mosquitoes. Findings argue for a limited role for vertical transmission for the perpetuation of CV virus as occurs with other related bunyaviruses. Issue: Five arboviruses have been previously reported from North Dakota mosquitoes. Two of these viruses caused significant epidemics and epizootics in humans and in horses. Western equine encephalomyelitis virus infected 1,180 humans and caused 96 deaths in 1941. Sixty-one years later in 2002, West Nile virus (WNV) was introduced into North Dakota and caused disease in 17 humans and 577 horses followed by 617 human cases, five human deaths, and 42 equine cases during 2003. Since then, North Dakota has reported human WNV cases every year and has one of the highest incident rates of neuroinvasive disease in the country. Response: To investigate arbovirus transmission in North Dakota, we collected mosquitoes from this region and screened them for viral infection by Vero cell culture assay. Seven viruses were isolated from 13 mosquito species. Spatial and temporal distributions of the important vectors of WNV, Cache Valley virus, Jamestown Canyon virus (JCV), and trivittatus virus are reported. Snowshoe hare virus, Potosi virus, and western equine encephalomyelitis virus were also isolated. The risks of Culex tarsalis and Aedes vexans transmitting WNV to humans were 62.24 % and 27.45% respectively, but in 2003 when the largest epidemic was reported, risks for Ae. vexans and Cx. tarsalis in Cass County were 71.73% and 26.05%, respectively. Risk of humans acquiring an infectious bite was greatest from mid-July through mid-August when the weekly vector index for Cx. tarsalis was greater than that of Ae. vexans. West Nile virus sequences were of the WN02 genotype. Most JCV strains belonged to a single clade of genetically related strains. Cache Valley virus and JCV were prevalent during August and early September and during July and August, respectively.Impact: Seven species of viruses circulated among 13 species of North Dakota mosquitoes during 2003-2006 and five of these viruses cause human disease. Culex tarsalis and Ae. vexans were both important vectors of WNV in North Dakota. This virus was isolated from nine species of mosquitoes and multiple isolations were made from four species, but Cx. tarsalis and Ae. vexans accounted for 90% of the total risk for the years 2003-2006. The relatively highinfection ratefor Cx. tarsalis and the abundance of Ae. vexans accounted for their relatively high risks. Cx. tarsalis was the more important with a risk factor of 62.24%. However, the largest epidemic of WNV infection in North Dakota was recorded in 2003, and, in Cass County with 32 cases, humans had a greater risk of being bitten by infected Ae. vexans.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Andreadis TG, Armstrong PM, Anderson JF, and Main AJ (2014) Spatial-temporal analysis of Cache Valley virus infection in Anopheline and Culicine mosquitoes in the northeastern United States, 1997-2012. Vector-Borne and Zoonotic Dis 14(10): 763-73.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Anderson JF, Main AJ, Armstrong PM, Andreadis TG, and Ferrandino FJ (In press) Arboviruses in North Dakota, 2003-2006. J Med Entomol.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Nelson R, Ciesielski T, Andreadis T, and Armstrong P (2014) Human case of eastern equine encephalitis- Connecticut, 2013. Connecticut Epidemiologist 34(3): 9-10.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Nelson R, Esponda B, Andreadis T, and Armstrong P (2014) West Nile virus Connecticut, 2000-2013. Connecticut Epidemiologist 34(2):5-7.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audiences include: medical entomologists, virologists, mosquito control personnel, federal, state and local public health officials. Efforts to deliver science-based knowledge to stakeholders included publication of results in peer-reviewed scientific journals (Journal ofMedical Entomology,New England Journal of Medicine, Proceedings Royal Society, London and Vector-Borne and Zoonotic Diseases) that have world-wide distribution to infectious disease specialists, medical entomologists and public health officials. Oral presentations were given at regional, national and international scientific conferences of vector-borne disease mosquito control organizations, and in interviews with local newspaper, radio and television reporters from Connecticut. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Results have been published in two peer-reviewed scientific journals Journal of Invertebrate Pathology and Vector-Borne Zoonotic Diseases, and presented at two scientific conferences; the 62nd Annual Meeting of the American Society for Tropical Medicine and Hygeine and the 59th Annual Meeting of the Northeastern Mosquito Control Association for dissemination to the scientific and mosquito control communities. What do you plan to do during the next reporting period to accomplish the goals? We will focus our research onthe underlying factors associated with the introduction, amplification, persistence, and range expansion of EEE virus in the norhteastern US including 1.) vector mosquito abundance and distribution that drive viral amplification and spillover into human and equine populations, 2) species-specific mosquito-avian interactions that favor amplification, 3.) virus titers in primary and secondary mosquito vectors, and 4) genetic variation in regional EEE virus strains that may provide evidence for local overwintering, evolution and extinction of EEE virus strains, andperiodic reintroduction from southern sources.
Impacts
What was accomplished under these goals?
Objective 1. Issue: Eastern Equine Encephalitis (EEE) virus is a highly pathogenic mosquito-borne agent responsible for periodic outbreaks of severe disease in humans and equines, causing high mortality and severe neurologic impairment in most survivors. During the last decade, episodes of EEE virus have reemerged in the northeastern US, where there has been increased virus activity and recurrent human and equine cases. These episodes occur when ecological conditions favor virus amplification followed by overflow into human and equine populations. EEE virus is maintained in an enzootic cycle involving the ornithophilic mosquito, Culiseta melanura and passerine birds in freshwater swamp habitats. Response: To evaluate the role of Cs. melanura and Culiseta morsitansin recent episodes of EEE virus activity in the region, we collected blood-fed mosquitoes between June, 2007, and October, 2008, from virus foci in 6 Massachusetts counties, and identified the source of blood meals by PCR amplification of mitochondrial cytochrome b gene and sequencing. Analysis of 529 Cs. melanura and 25 Cs. morsitans revealed that nearly 99% and 96% of mosquitoes, respectively, acquired blood meals solely from avian hosts. American Robin, Turdus migratorius was identified as the most common vertebrate host for Cs. melanura (21.7%, n = 115), followed by Tufted Titmouse, Baeolophus bicolor (8.7%, n = 46), Black-capped Chickadee, Poecile atricapillus (8.5%, n = 45), Scarlet Tanager, Piranga olivacea(6.8%, n = 36), Field Sparrow, Spizella pusilla(6.2%, n = 33), Northern Cardinal, Cardinalis cardinalis(5.7%, n = 30), and other mostly Passeriformes birds. Mammalian-derived blood meals were identified as white-tailed deer, Odocoileus virginianus, domestic cow, Bos taurus,and human, Homo sapiens. Impact: Our results in conjunction with other lines of evidence, including reservoir competency, prevalence of antibody, and infection in nature, suggest that the American Robin, Tufted Titmouse, Black-capped Chickadee, and a few other passerine birds may play key roles in supporting EEE virus transmission in this region. Infrequent blood feeding of Cs. melanura on mammalian hosts, including humans, also indicates that this mosquito may occasionally contribute to epidemic/epizootic transmission of EEE virus in the region. Objective 3. Issue: Ochlerotatus japonicusis an invasive rock hole and container-breeding mosquito native to East Asia that was first detected in the northeastern US in 1998. It has rapidly spread throughout much of eastern North America where it is now firmly established, and appears to be a dominant invader that may be competitively displacing native species in natural rock hole and used tire habitats. More recently, the species has invaded Canada and central Europe.It is presumed to have entered both the US and Europe through the international trade in used automobile tires. The establishment of the species in these regions of the world is of considerable public health significance.It is an aggressive human biter, is a competent vector of several important arboviruses including EEE, Japanese encephalitis, LaCrosse virus and St. Louis encephalitis, and has been incriminated in transmission of West Nile virus in North America. When an exotic species is introduced into a new region in the absence of any natural enemies that attack it in its native range, the likelihood of successful establishment and expansion of the invading species can be appreciably enhanced provided that species is able to cope with existing environmental conditions, effectively compete with resident species that occupy the same or similar ecological niche, and is not beset by native parasites or predators. This phenomenon has been described as an “enemy release hypothesis” wherein invading organisms lose their co-evolved parasites and other natural enemies during the process of invasion leading to higher demographic success which in turn may lead to a competitive advantage for the invader over native species. Accordingly, surveys of North American populations of Oc. japonicus have yet to uncover any significant parasites or predators. Response: In 1980, a microsporidian parasite was discovered infecting Oc. japonicus larvae collected from rock pools along the Okudake River, in the Oita Prefecture, Kyushu Region of Japan but was never formally described. In 2010, the original collection site in Japan was revisited in an attempt to recover this microsporidium and evaluate its potential as a biological control agent for possible introduction into North American populations of Oc. japonicus.The microsporidium was again found infecting larval populations of Oc. j. japonicus and Oc. hatorii, successfully isolated and sent to our laboratory for analyses that included: complete morphological characterization of the life cycle by light and electron microscopy, an elucidation of transmission pathways andexamination of SSU rDNA sequence data to determine its unique placement among other mosquito-parasitic microsporidia.The microsporidium was found to be both vertically and horizontally transmitted, exhibit dimorphic development alternating between diplokaryotic and monokaryotic stages and produce two morphologically distinct spores, one in larvae and another in adult females.Horizontal transmission of infection to larval mosquitoes occured via direct oral ingestion of uninucleate spores that were produced in vertically-infected larval hosts. Development in horizontally-infected hosts was diplokaryotic following karyokinesis of uninucleate schizonts and binary fission to produce small (4.3 mm x 2.0 mm) membrane free, ovoid, binucleate spores that are confined to adult female reproductive tissues (ovariole sheath and oviducts). Vertical transmission of the microsporidium from adult females to larval progeny took place via surface contamination of the egg (transovum).Microsporidian development in vertically-infected larvaewas haplophasic with unpaired nuclei throughout, producing rosette-shaped sporogonial plasmodia contained within a thin non-persistent sporophorous vesicle and culminating in the formation of membrane free, uninucleate, conical spores (7.0 µm x 2.8 µm).Developmentwas confined to host fat body tissue which appeared as swollen white masses in the thorax and selected segments of the abdomen causing larvae to appear abnormally distorted and results in death during the third and fourth instar stages.The SSU rDNA sequences obtained from the two morphologically identical microsporidia isolated from Oc. japonicus and Oc. hatorii were nearly identical and unique when compared with GenBank entries of all other mosquito-parasitic species.Phylogenetic trees constructed by Maximum Parsimony, Maximum Likelihood and bootstrap analyses using the Neighbor Joining search parameter yielded similar typologies. In each case, the novel microsporidium was the sister group to the clade containing Parathelohania species from Anopheles mosquitoes and the monotypic Novothelohania ovalae from Ochlerotatus caspius showing approximately 10% to 13% sequence divergence to those two genera providing strong support for establishment as a separate genus. Impact: A new genus and species of Microsporidia, Takaokaspora nipponicus n. gen., n. sp. was described and fully characterized from the invasive exotic mosquito, Oc. japonicus based on light microscope and ultrastructural morphology, developmental features, transmission cycles and comparative sequence analyses of the small subunit ribosomal DNA (SSU rDNA).This essential information on the biology of this parasite will now make it possible to fully evaluate its potential as a biological control agent for possible introduction into North American populations of Oc. japonicus.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Andreadis, T. G., Takaoka, H., Otsuka, Y., and Vossbrinck, C. R. 2013. Morphological and molecular characterization of a microsporidian parasite, Takaokaspora nipponicus n. gen., n. sp. from the invasive rock pool mosquito, Ochlerotatus japonicus japonicus. J. Invertebr. Pathol. 114:161-172.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Armstrong, P. M. and Andreadis, T. G. 2013. Eastern equine encephalitis virus: an old enemy but a new threat. NE. J. Med. 368:1670-1673.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Armstrong, P. M., Anderson, J. F., Farajollahi, A. Healy, S. P., Unlu, I., Crepeau, T. N. Gaugler, R., Dina M. Fonseca, D. M., and Andreadis, T. G. 2013. Isolations of Cache Valley virus from Aedes albopictus (Diptera: Culicidae) in New Jersey and evaluation of its role as a regional arbovirus vector J. Med. Entomol. 50:1310-1314.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Huang, S., Smith, D. J., Molaei, G., Andreadis, T. G., Larsen, S. E., and Luccchesi, E. F. 2013. Prevalence of Dirofilaria immitis (Spirurida: Onchocercidae) infection in Aedes, Culex and Culiseta mosquitoes from North San Joaquin Valley, CA. J. Med. Entomol. 50:1315-1323.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Molaei, G., Andreadis, T. G., Armstrong, P. M., Thomas, M. C., Deschamps, T., Cuebas-Incle, E., Montgomery, W., Osborne, M., Smole, S., Matton, P., Andrews, W., Best, C., Cornine III, F., Bidlack, E., and Texeira, T. 2013. Vector-host interactions and epizootiology of eastern equine encephalitis virus in Massachusetts, USA. Vector-Borne Zoonotic Dis. 13:312-323.
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2014
Citation:
Becnel, J. J. and Andreadis T. G. 2014. Microsporidia in insects. In: M. Witter and J. J Becnel (eds.), Microsporidia: Pathogens of Opportunity. John Wiley & Sons, Inc., Hoboken, NJ. (in press)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Andreadis, T. G. Global climate change and mosquito-borne diseases. Proc. 58th Ann. Mtg. Northeastern Mosquito Control Association, Mystic, CT, December 2012
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The overwintering ecology of Culiseta melanura (Coquillett) was studied in a seasonally flooded evergreen forest swamp in south central Connecticut in an effort to clarify which larval stages successfully overwinter in the northeastern United States, and to determine the degree to which larval development and/or mortality occur during the winter months. A total of 8,626 immature Cs. melanura were collected weekly for analysis from subterranean crypts and cavities located under the roots of trees from December 13, 2011 to May 31, 2012. Despite the formation of ice on the surface water at the entrance holes to the crypts, water temperatures within the cavities remained above freezing (ave. = 1.8C) throughout coldest winter months of January and February. A heterogeneous population of 2nd, 3rd and 4th instar larvae were recovered throughout the winter and early spring in the same relative proportions (30%, 30%, 40%, respectively) with no significant change in their comparative abundance during this period providing unequivocal evidence that all three instars successfully overwinter in the region. Findings further demonstrate that larvae undergo no development during the winter and do not appear to be impacted by any measurable mortality. The cessation of larval diapause and a resumption of development were observed in mid-April and were coincident with a gradual increase in water temperature within the crypts to 9C, in agreement with a previously calculated developmental thermal minimum of 8.5C for Cs. melanura. This resulted in a protracted period of pupation that encompassed a minimum of five weeks, followed by a staggered emergence of adults and an overlap of the residual overwintering population with larvae of the first summer generation. Disease outbreaks caused by eastern equine encephalitis virus (EEEV; Togaviridae, Alphavirus) may be prevented by implementing effective surveillance and intervention strategies directed against the mosquito vector. Methods for EEEV detection in mosquitoes include a real-time reverse transcriptase PCR technique (TaqMan assay), but we report its failure to detect variants isolated in Connecticut in 2011, due to a single base-pair mismatch in the probe-binding site. To improve the molecular detection of EEEV, we developed a multi-target TaqMan assay by adding a second primer/probe set to provide redundant targets for EEEV detection. The multi-target TaqMan assay had similar performance characteristics to the conventional assay but also detected newly evolving strains of EEEV. The approach increases the reliability of TaqMan assay by creating backup targets for virus detection without sacrificing sensitivity or specificity. PARTICIPANTS: There were two main investigators on this project, Theodore G. Andreadis (Chief Medical Entomologist) and Philip M. Armstrong (Associate Virologist) from the Center for Vector Biology & Zoonotic Diseases at The Connecticut Agricultural Experiment Station. Dr. Andreadis coordinated the weekly collection and identification of mosquitoes from field sites and analyzed the data. Dr. Armstrong developed and analyzed new primer/probe sets to identify EEEV isolates from mosquito pools and Vero cell cultures. John Shepard and Michael Thomas of The Connecticut Agricultural Experiment Station provided technical assistance in collecting, processing and identifying mosquitoes. Nicholanna Prince, Connecticut Department of Public Health, assisted in initial identification and development of the new primer/probe sets. TARGET AUDIENCES: The target audiences are as follows: medical entomologists, virologists, mosquito control personnel, federal, state and local public health officials. Efforts to deliver science-based knowledge to stakeholders included publication of results in peer-reviewed scientific journals (Journal of the American Mosquito Control Association Emerging and Vector-Borne and Zoonotic Diseases) that have world-wide distribution to infectious disease specialists, medical entomologists and public health officials. Oral presentations were given at regional, national and international scientific conferences of vector-borne disease mosquito control organizations, and in interviews with local newspaper, radio and television reporters from Connecticut. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
With this investigation, we provide unequivocal evidence that in freshwater swamp habitats in this region of the northeastern US, a heterogeneous population of 2nd through 4th instar Cs. melanura larvae successfully overwinter in subterranean crypts beneath the roots of trees where water temperatures remain above freezing throughout the winter months. The recovery of large numbers of all three instar stages in the same relative proportions throughout the winter, with no discernible change in their comparative abundance, further lead us to conclude that larvae undergo no development during this period and are not severely impacted by any measurable mortality. Our observations on the renewal of larval development when water temperatures attained 9C validate a heat summation model that calculated a developmental thermal minimum of 8.5C for Cs. melanura. This minimum developmental temperature was further substantiated by the lack of any discernible development within the larval population during late March and early April when water temperatures of 7C were consistently recorded in the crypts for three straight weeks. Findings further support a degree-day model which predicts that temperature driven development of 1st through 4th overwintering instar Cs. melanura larvae creates a cascade of adult emergence peaks among the first generation in the spring and early summer based on the degree-days required for completion of each stadium. Findings provide new insights and how warming winter temperatures associated with climate variability may impact the maintenance and amplification of EEEV in the region. TaqMan Assays may be combined to detect multiple targets of the same pathogen as shown in this study or to detect multiple pathogens in multiplex reactions. This flexibility allows one to rapidly extend assay coverage when a new virus or virus variant emerges in an area. We accomplished this by adding our in-house primer/probe set to a previously published TaqMan assay with no further modification. The resulting assay was equally sensitive to the single-target assay, specific for North American strains of EEEV, and could be used in mosquito pools. Our multi-target assay provides an expedient solution to account for newly emergent variants of EEEV and this approach could be applied to the molecular diagnostics of other rapidly evolving viruses. In the meantime, it will be important to monitor the continued efficacy of molecular assays for their viral targets and consider combining assays when appropriate. If mutations arise too frequently or become fixed within target regions, then primer and probes will need to be completely redesigned to ensure adequate coverage for contemporaneous virus strains
Publications
- Hardstone, M. C. and Andreadis, T. G. 2012. Weak larval competition between the invasive mosquito, Aedes japonicus japonicus (Diptera: Culicidae) and three resident container-inhabiting mosquitoes under standard laboratory conditions. J. Med. Entomol. 49:277-285.
- Molaei, G., Huang, S., and Andreadis, T. G. 2012. Vector-host interactions of Culex pipiens complex mosquitoes in northeastern and southern USA. J. Amer. Mosq. Control Assoc. 28:127-136
- Morningstar, R. J., Hamer, G. L., Goldberg, T. L., Huang, S., Andreadis, T. G., and Walker, E. D. 2012. Diversity of Wolbachia pipientis strain wPip in a genetically admixtured, above-ground Culex pipiens (Diptera: Culicidae) population: association with form molestus ancestry and host selection patterns. J. Med. Entomol. 49:474-481.
- Andreadis, T. G. 2012. The contribution of Culex pipiens complex mosquitoes to transmission and persistence of West Nile virus in North America. J. Amer. Mosq. Control Assoc. 28s:137-151.
- Andreadis, T. G. and Armstrong, P. M. 2012. 2012: a record year for West Nile virus activity in Connecticut. CT Weekly Agric. Report 92:1-4.
- Andreadis, T. G., Shepard, J. J. and Thomas, M. C. 2012. Field observations on the overwintering ecology of Culiseta melanura in the northeastern United States. J. Amer. Mosq. Control Assoc. 28:286-291.
- Andreadis, T. G., Simakova, A. V., Vossbrinck, C. R, Shepard, J. J., Yurchenko, Y. A. 2012. Ultrastructural characterization and comparative phylogenetic analysis of new Microsporidia from Siberian mosquitoes: evidence for coevolution and host switching. J. Invertebr. Pathol. 109:59-75.
- Armstrong, P. M., Prince, N. and Andreadis, T. G. 2012. Development of a multi-target TaqMan assay to detect eastern equine encephalitis virus variants in mosquitoes. Vector-Borne Zoonotic Dis. 12:872-876.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: West Nile virus (WNV) has become firmly established in northeastern US, reemerging every summer since its introduction into North America in 1999. To determine whether WNV overwinters locally or is reseeded annually, the patterns of viral lineage persistence and replacement in Connecticut were examined over 10 consecutive transmission seasons by phylogenetic analysis. We also compared the full protein coding sequence among WNV isolates to search for evidence of convergent and adaptive evolution. Viruses sampled from Connecticut segregated into a number of well-supported subclades by year of isolation with few clades persisting ≥ 2 years. Similar viral strains were dispersed in different locations across the state and divergent strains appeared within a single location during a single transmission season, implying widespread movement and rapid colonization of virus. Numerous amino acid substitutions arose in the population but only one change, V to A at position 159 of the envelope protein, became permanently fixed. Several instances of parallel evolution were identified in independent lineages, including one amino acid change in the NS4A protein that appears to be positively selected. Results suggest that annual reemergence of WNV is driven by both reintroduction and local-overwintering of virus. Despite ongoing diversification of WNV, most amino acid variants occurred at low frequencies and were transient in the virus population. Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile, dengue and chickungunya viruses. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. We describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing ≤50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures. Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (WNV, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population. Collaborations with scientists from the University of New Mexico and Broad Institute at the Massachusetts Institute of Technology and Harvard University contributed greatly to research outputs and impacts. PARTICIPANTS: There were several investigators on this project, Philip M. Armstrong, Theodore G. Andreadis, John F. Anderson and Charles R. Vossbrinck (Center for Vector Biology & Zoonotic Diseases, The Connecticut Agricultural Experiment Station); Gregory D. Ebel and Kendra N. Pesko (Department of Pathology, University of New Mexico School of Medicine); and Bruce W. Birren, Mathew R. Henn, Niall J. Lennon, and Ruchi M. Newman (Broad Institute of Massachusetts Institute of Technology and Harvard). Dr. Armstrong conducted the virus isolation and identification and analyzed and evaluated the data, Dr. Andreadis coordinated the collection and identification of mosquitoes, Dr. Anderson assisted in the isolation and indentification of virus isolates, Dr. Vossbrinck assisted in the phylogenetic analysis, and Drs. Ebel, Pesko, Birren, Henn, Lennon, and Newman assisted in the virus sequencing. Shannon Finan, John Shepard, and Michael Thomas of The Connecticut Agricultural Experiment Station provided technical assistance in processing and identifying mosquitoes and isolating virus in Vero cell culture. TARGET AUDIENCES: The target audiences are as follows: medical entomologists, virologists, mosquito control personnel, federal, state and local public health officials. Efforts to deliver science-based knowledge to stakeholders included publication of results in a peer-reviewed scientific journal, Virology that has world-wide distribution to infectious disease specialists, medical entomologists and public health officials, oral presentations at regional and national scientific conferences of mosquito control organizations, and in interviews with local newspaper, radio and television reporters in Connecticut. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our analysis describes the patterns of viral lineage turnover and protein evolution within a region supporting stable WNV transmission. We observed evidence of local overwintering of virus but without permanent establishment of local populations. Moreover, we documented the monthly and yearly appearance of distinct variants, implying rapid re-colonization of virus in a given locale. Numerous nucleotide changes have arisen since its introduction into North America, but negative selection appeared to constrain changes at the protein level. Finally, we identified several instances of convergent evolution, including one amino acid change that appears to be positively selected. These investigations will allow scientists to monitor and tract local regional changes in WN virus that may be associated with changes in virulence and/or transmission efficiency as it evolves over the years. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population. Results were published in Virology, a peer-reviewed journal that has international distribution, and the Journal of Visualized Experiments, a web-based journal dedicated to accelerating biological, medical, chemical and physical research by elucidating techniques through a combination of peer reviewed video and text.
Publications
- Anderson, J.F., F.J. Ferrandino, D.W. Dingman, A.J. Main, T.G. Andreadis and J.J. Becnel. 2011. Control of mosquitoes in catch basins in Connecticut with Bacillus thuringiensis israelensis, Bacillus sphearicus and spinosid. J. Am. Mosq. Control Assoc. 27:45-55. Armstrong, P.M, T.G. Andreadis, S. Finan, J.J. Shepard, M.C. Thomas and J.F. Anderson. 2011. Detection of infectious virus from field-collected mosquitoes by Vero cell culture assay. J. Visualized Exper. 52. http://www.jove.com/index/Details.stpID=2889,doi: 10.3791/2889.
- Armstrong, P.M., C.R. Vossbrinck, T.G. Andreadis, J.F. Anderson, K.N. Pesk, R.M. Newman, N.J. Lennon, B.W. Birren, G.D. Ebel and M.R. Henn. 2011. Molecular evolution of West Nile virus in a northern temperate region: Connecticut, USA 1999-2008. Virol. 417:203-210.
- Huang, S., G. Molaei and T.G. Andreadis. 2011. Reexamination of Culex pipiens hybridization zone in the eastern United States by ribosomal DNA-based single nucleotide polymorphism markers. Am. J. Trop. Med. Hyg. 85:434-441.
- Simpson, J.E., P.J. Hurtado, J. Medlock, G. Molaei, T.G. Andreadis, A.P. Galvani and M.A. Diuk-Wasser. 2011. Vector host-feeding preferences drive transmission of multi-host pathogens: West Nile virus as a model system. Proc. R. Soc. B. doi:10.1098/rspb.2011.1282.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: During the last 6 years, eastern equine encephalitis virus (EEEV) has reemerged in the northeastern US, resulting in 26 human cases and 9 fatalities. Virus transmission has intensified throughout this region and spread to locales where it was not previously detected. Disease outbreaks caused by EEEV strike at irregular intervals when ecological conditions favor virus amplification and overflow into human and equine populations. EEEV is perpetuated in an enzootic cycle involving ornithophilic mosquitoes (primarily Culiseta melanura) and passerine birds in freshwater swamps. Human and equine cases occur infrequently despite relatively high rates of EEEV infection in Cs. melanura during virus amplification. Other mosquito species such as Aedes vexans, Coquillettidia perturbans, Ochlerotatus canadensis, and Oc. sollicitans have been implicated as epidemic/epizootic bridge vectors. Although Cs. melanura feeds infrequently on mammals, its ability to serve as a bridge vector may be offset by a much higher prevalence of EEEV infection in this species. One of the criteria used for incriminating enzootic and bridge vectors is based on the frequency of virus detection from each species. Typically, mosquitoes are collected from endemic sites and screened for the presence of virus by cell culture and/or molecular methods. This procedure provides critical information on the identity, spatial and temporal distribution, and proportion of virus-infected mosquitoes, and forms the basis for many arbovirus surveillance programs. However, virus titers may vary considerably within infected mosquitoes reflecting their ability to support virus replication which is a necessary precondition for mosquitoes becoming infectious. In 2009, we experienced a substantial increase in EEEV activity in Connecticut, with numerous virus isolations from Cs. melanura and potential bridge vectors. To evaluate their capacity to replicate and transmit virus, we estimated the infection prevalence and virus titers in mosquitoes by cell culture, plaque titration, and quantitative RT-PCR with the expectation that the most efficient vectors will support consistently high virus titers. A total of 291,641 mosquitoes (35 species) were collected and processed as 16,909 pools for virus isolation. EEEV was isolated from 122 mosquito pools representing 14 different species and 7 genera. Cs. melanura yielded the greatest number of EEEV isolations (n=83), followed by Oc. canadensis (n=10) and Ae. cinereus (n=6). Relatively few (less than 4) or no EEEV isolates were obtained from the remaining mosquito species collected. Mosquito pools that yielded EEEV in cell culture were directly tested by qRT-PCR. A total of 108 mosquito pools yielded positive Ct values. Mean Ct values were lowest for Cs. melanura (Ct=22.3) and exceeded 30 for all other mosquito species, suggesting species-specific differences in virus titer. The concentration of infectious virus was estimated from positive mosquito pools by plaque titration in Vero cell culture. Cs. melanura had significantly higher virus titers (mean=6.5 log10 plaque forming units (PFU)/mosquito pool) than all other mosquito species (2.9-0.8 PFU). PARTICIPANTS: There were two main investigators on this project, Philip M. Armstrong (Associate Virologist) and Theodore G. Andreadis (Chief Medical Entomologist) from the Center for Vector Biology & Zoonotic Diseases at The Connecticut Agricultural Experiment Station). Dr. Andreadis coordinated the collection and identification of mosquitoes from established field sites. Dr. Armstrong conducted virus isolation in Vero cells, identification by RT-PCR, and quantification by plaque assay. Shannon Finan, John Shepard, and Michael Thomas of The Connecticut Agricultural Experiment Station provided technical assistance in processing and identifying mosquitoes and isolating virus in Vero cell culture. TARGET AUDIENCES: The target audiences are as follows: medical entomologists, virologists, mosquito control personnel, federal, state and local public health officials. Efforts to deliver science-based knowledge to stakeholders included publication of results in a peer-reviewed scientific journal, Emerging Infectious Diseases that has world-wide distribution to infectious disease specialists, medical entomologists and public health officials, oral presentations at regional and national scientific conferences of mosquito control organizations, and in interviews with local newspaper, radio and television reporters in Connecticut. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our analysis of EEEV-positive mosquito pools revealed striking differences in virus titer among different mosquito species collected in Connecticut. Cs. melanura was the only species that developed titers associated with EEEV transmission, previously estimated between 4-7 log10 PFU of virus in transmitting mosquitoes. This suggests that EEEV is transmitted primarily by Cs. melanura in this region of the northeastern US despite repeated virus isolations from other mosquito species. Infrequent human and horse cases may arise when Cs. melanura occasionally feeds on mammals rather than by participation of another epidemic/epizootic bridge vector. Prior studies identified mammalian-derived blood meals in 1-10% of Cs. melanura collected from Massachusetts, Connecticut, and New York providing a direct conduit for virus transmission by this species to horses and humans. These analyses, in conjunction with observations on vector longevity, vector competence, and prevalence of EEEV infection in Cs. melanura, suggest that this species could serve as both enzootic and epidemic/epizootic bridge vector of EEEV. We provide additional support for this hypothesis by estimating virus titers in field-collected mosquitoes, allowing us to determine which infected mosquitoes could possibly transmit virus. Findings highlight the importance of considering virus titer when interpreting virus isolation or PCR-detection data from field-collected mosquitoes. Although we isolated EEEV from a number of mosquito species, Cs. melanura was the only species to produce consistently high titers of EEEV sufficient for transmission. This finding may help reconcile the paucity of symptomatic human and equine cases, despite frequent detection and/or isolation of virus from mammalophilic mosquitoes during episodes of virus amplification. These results should be verified in other geographic regions, where the involvement of other locally abundant mosquitoes is suspected during disease outbreaks. By incorporating information on virus titers in mosquitoes, the number of candidate vectors may be narrowed down to a few key species that are capable of supporting virus transmission in nature.
Publications
- Andreadis, T. G. and Wolfe, R. J. 2010. Evidence for reduction of native mosquitoes with increased expansion of the invasive Ochlerotatus japonicus japonicus (Diptera: Culicidae) in the northeastern United States. J. Med. Entomol. 43-52.
- Andreadis, T. G., Armstrong, P. A., and Bajwa, W. J. 2010. Studies on hibernating populations of Culex pipiens (Diptera: Culicidae) from a West Nile virus endemic focus in New York City: parity rates and isolation of West Nile virus. J. Am. Mosq. Control Assoc. 26:257-264.
- Armstrong, P. A. and Andreadis, T. G. 2010. Eastern equine encephalitis virus in mosquitoes and their role as bridge vectors. Emerging Inf. Dis. 16:1869-1874.
- Diuk-Wasser, M. A., Molaei, G., Simpson, J. E., Folsom-OKeefe, C. M., Armstrong, P. M., and Andreadis, T. G. 2010. Avian communal roosts as amplification foci for West Nile virus in urban areas in northeastern United States. Am. J. Trop. Med Hyg. 82:337-343.
- Molaei, G., Cummings, R. F., Su, T., Armstrong, P. M., Williams, G. A., Cheng, M. L., Webb, J. P., and Andreadis, T. G. 2010. Vector-host interactions governing epidemiology of West Nile virus in southern California. Am. J. Trop. Med. Hyg. 83:1269-1282.
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