THE EFFECT OF ECOLOGICAL AND ENVIRONMENTAL FACTORS ON WEST NILE VIRUS PREVALENCE IN HUMANS Matthew Schell BIOS 3010 Ecology, fall 2012 Department of Biological Sciences, Western Michigan University Kalamazoo, MI 49008, USA Abstract This study set out to investigate the ecological factors which influence the prevalence of West Nile Virus in humans. Several different ecological variables were examined including: temperature, precipitation, urbanization, hydrogeography, association with birds, and vector species feeding patterns. The extent to which these variables affect the abundance of the vector mosquito species population and thus, infection rates in humans is varied. An increase in temperature had the strongest and most consistent effect on increasing abundance of mosquitoes and infection rates in humans. Increased urbanization and decreased hydrogeography also increased human infection rates. Results for precipitation were inconclusive, as were correlations with vector species association with birds and feeding patterns. These variables all contribute significantly to modeling both mosquito populations and infection rates in humans, and continued studies examining the interplay of these variables will result in models with increasing accuracy. Key words: West Nile Virus, mosquito, vector-­‐borne disease, climate, ecology, corvid birds, temperature, precipitation, urbanization, hydrogeography Introduction West Nile Virus (WNV) is a vector born disease that was first found in the United States in 1999, having originated from Uganda. West Nile Virus is now a large public health concern near the end of every summer because of the prevalence of effective mosquito vectors at this time of year. Generally, WNV causes asymptomatic illness in humans, with some people developing flu-­‐like symptoms that last for a few days. In rare cases WNV can progress to encephalitis, which can lead to permanent brain damage or death (Goldblum et al. 1956). The virus was first successfully isolated in Uganda in 1937 from a woman in the West Nile district in Northern Uganda (Smithburn et al. 1940). The initial tests which were done on the virus revealed that WNV was similar in nature to other vector borne diseases capable of causing encephalitis such as St. Louis encephalitis, louping ill, and Japanese B encephalitis (Smithburn et al. 1940). After this first isolation, the virus remained common and at endemic levels throughout Africa with occasional forays into the Middle East and Africa (Tsai et al. 1998). Periodic epidemics occurred in Israel and Africa throughout this time period; however, Europe remained relatively free of the virus until 1996 when the first epidemic of WNV struck Southeastern Romania (Tsai et al. 1998). The first major outbreak in Europe was closely followed by the transfer of the virus to North America in 1999. The virus was first isolated in New York in the late summer of that year (Lanciotti et al. 1999). Using DNA evidence, it was determined that the strain of WNV present in New York was most closely related to a strain of WNV isolated from Israel in 1998 (Lanciotti et al. 1998) (Figure 1). This allows a clearer picture of how the introduction of WNV to North America occurred. Three explanations seem plausible. First, an individual may have contracted WNV while in Israel and returned to the United States. After returning to the United States that individual may have been bitten by a mosquito while still infected. The now infected mosquito may have then fed on other hosts, thus transmitting and amplifying the virus. Second, some sort of infected animal host may have been inadvertently transported to New York via airplane or ship. Third, one or more infected mosquitoes may vectors have been transported and upon arrival in the United States, these mosquitoes could have then infected a number of hosts, amplifying and transmitting the virus. After arriving in the United States, WVN quickly spread throughout the continental US, moving westward with each successive year and was present in all areas of the United States by 2008 (Young and Jensen 2012). It has been proposed that the spreading mechanism for WNV was along bird migratory routes, since normal mosquito dispersal patterns were not extensive enough to account for the rapid spread of the virus (Young and Jensen 2012). The distribution of WNV cases throughout the US, however, is not uniform, but aggregated in certain areas. Generally, there appears to be more cases of WNV in and near urban centers. This is true when looking at absolute numbers of human WNV cases, but also when looking at proportions. In addition to the aggregation of cases near urban areas, a much higher than expected proportion of human cases is found in the upper mid-­‐west and the Mississippi River Valley (Young and Jensen 2012) (Figure 2 and Figure 3). As with any emerging disease, WNV is a cause for concern because of the unknown effects it will have on human and animal populations throughout North America. While data exists on the prevalence and intensity of infections in other parts of the world, ecological and environmental factors in the United States are different and may lead to a higher incidence of infection. In addition, WNV has shown the ability to form new strains, as evidenced by the extensive phylogenic tree stretching back to the initial isolation (Lanciotti et al. 1999). It is possible that a new strain may become more pathogenically virulent than previous strains, thus leading to a higher incidence of deaths or serious health problems among humans. Also, the possibility of a new public health issue has the potential to add a great amount of cost to the health care system in America. All of these valid concerns about WNV have led to extensive research in an effort to learn about and control the spread of the disease. Considering the potential effects of extensive WNV infection among humans, understanding the way in which WNV is spread is of the utmost importance to medical personnel. Ideally, the basic reproductive rate (Rp) of WNV would be lower than 1, leading to a decrease in the number of human cases experienced by the population each year. Since the reproductive rate is a product of disease latency, density of susceptibles, and transmission rate, where Rp=LSβ, there are two ways in which it is possible to lower the Rp of WNV (Begon et al. 2006). First, the transmission rate (β) of the disease could be lowered. In the context of WNV this would mean reducing the probability that an infected mosquito comes in contact with a susceptible human, or reducing the affinity of infected mosquitoes for human hosts. To reduce β the number of infected mosquitoes would have to be reduced, meaning that control measures for mosquito populations in WNV-­‐prone areas would need to be instituted. Decreasing Rp may also be accomplished by reducing the number of susceptible human hosts, S, in the population. The discovery and use of a vaccine for WNV would be a possible way to reduce the number of susceptible individuals. In addition to reducing the number of susceptible individuals, a vaccine may result in decreased length of infectious periods, L, in individuals who experience secondary breakthrough of the disease (Andre et al. 2008). Those individuals who receive a vaccination, yet still contract the disease generally have more mild cases for a shorter duration (Andre et al. 2008). However, in order for a vaccination program to be effective, a critical proportion of the population (pc) would need to be vaccinated in order to establish herd immunity. The proportion of a population required to reach herd immunity is related to Rp by the equation pc=1-­‐(1/Rp) (Begon et al. 2006). Normally the proportion of the population required to reach herd immunity is quite high, requiring a great deal of money and effort to be successful. While the development and widespread use of a vaccine would most likely be successful, at this point in time the number and severity of WNV cases do not create the demand for such efforts. Therefore, affecting β is a much more probable solution to the problem of human WNV infection when considering costs and benefits. In order to institute the most effective control measures, modeling mosquito populations in a region is important. The most effective control measures will be established in areas of high vector mosquito abundance, or areas of high WNV infection rates; therefore, the ability to predict these at-­‐risk areas using models is a useful tool. West Nile Virus is a vector born disease, transmitted by certain mosquito species as vectors; thus, controlling mosquito distribution and abundance will lead to a decrease in the number of human WNV infections. Many species of mosquitos are present in the United States; however, a few species have been identified as having the greatest impact on human WNV cases. These species include: Culex pipiens, Culex quinquefasciatus, and Culex restuans (Molaei et al. 2006; Hamer et al. 2008; Farajollahi et al. 2011). The distribution and abundance of these mosquitos, as with most other organisms, is a result of a combination of environmental and ecological factors. Many of these factors are useful for modeling mosquito populations and human WNV infection rates. Factors that influence mosquito vector abundance include temperature, precipitation, urbanization, hydrogeography, and host species abundance. It is important to note that many of the mosquito species that play host for WNV feed on diets of birds and mammals, with most showing a preference for birds. Therefore, the distribution and abundance of non-­‐human hosts, such as birds, may play a role in vector mosquito species distribution and abundance. Also, these environmental and ecological variables do not operate in a vacuum, but many times interact and influence each other. Therefore, while it is useful to examine each variable in an isolated way, looking at the big picture is equally important. If there was no relationship among human WNV cases, mosquito abundance, and ecological factors, then it would be expected that the incidence of human infections would randomly occur throughout any given area independent of mosquito abundance and ecological variables. A series of null hypotheses can be developed to examine these ecological factors. First, temperature will have no effect on mosquito vector density and WNV transmission. Precipitation will have no effect on mosquito vector density and WNV transmission. Urbanization will have no effect on mosquito density and WNV transmission. Hydrogeography will have no effect on mosquito vector density and WNV transmission. Finally, mosquito feeding patterns and density of avian hosts will have no effect on mosquito vector density and WNV transmission. Alternatively, if human WNV infections are affected by mosquito abundance and the ecological factors named in the null hypotheses, then aggregation of human infections will be evident. In order to determine the validity of the hypotheses several lines of inquiry must be investigated. It must be determined what variables affect the distribution and abundance of vector mosquito species (temperature, precipitation, urbanization, hydrogeography, mosquito feeding patterns, and density of avian hosts). Therefore, each variable that affects the mosquito species must be investigated and related to a change in mosquito abundance. Positive and negative effects on mosquito population will allow for a greater understanding of mosquito population dynamics. Feeding and behavioral patterns of the vector is important to understand in order to determine the extent to which infected mosquitoes are coming into contact with humans. A mosquito population with a high prevalence of infection, but low preference for human hosts may not be as dangerous as a population with a moderate rate of infection, and high preference for human hosts. Methods The idea for this review came from the extensive media coverage given to WNV at the time when I was choosing my topic. My research into WNV included searches on three different databases at Western Michigan University’s library: Web of Science, BIOSIS Previews, and Pubmed. The objective for my initial research into this topic was to build background knowledge, to understand the major areas on inquiry in WNV research, and what ecological concepts were being investigated. I spent 1.5 hours on this part of the research, using different key word searches in all 3 databases and gathering a wide array of sources. My first keyword search used three terms: “west nile”, “mosquito”, and “climate”, which returned results of 108 on Web of Science, 37 on BIOSIS Previews, and 94 on Pubmed. The second keyword search used: “west nile”, “temperature”, and “geography”, and returned 8 results on Web of Science, 0 on BIOSIS Previews, and 24 on Pubmed. The final keyword search of “west nile”, “mosquito”, and “geography” gave results of 23 on Web of Science, 6 on BIOSIS Previews, and 24 on Pubmed. From these initial searches I found 10 articles that I determined to be useful in my paper including: 8 from Web of Science, 1 from BIOSIS Previews, and 1 from Pubmed. After the initial search, using general keywords related to the topic of WNV, I continued to return to the databases for two reasons. First, I would search for more articles when a new path on inquiry was discovered. Second, if I was unable to draw conclusions about a specific variable, or I required more information on the topic, I would do more research. For example, as I was reading an article found in the initial searches, I came across a discussion of the correlation of corvid birds and WNV. Therefore, I returned to the databases and used the search terms “west nile” and “corvid birds” in order to obtain articles looking at the relationship between these two topics. More commonly, I searched for articles cited within the article I was reading. This seemed to be the most efficient way to find relevant articles. I noticed that while searching with key words many unrelated articles would be found, which would require time to sort through and identify relevant ones. In addition to the data collected, I noticed some differences in the types of articles I would find when comparing databases. Web of Science was by far the most useful database for my purpose. This was where I found the vast majority of my articles, and it was the first database I would go to when looking for a specific article cited in previously found literature. I noticed that BIOSIS Previews seemed to return a fewer number of articles using the same keywords than Web of Science, and many articles would be (were?) the same. While Pubmed contained a great deal of articles related to the medical implications and descriptions of WNV, few were related to ecological issues affecting the spread of the infection. Therefore, I rarely found myself returning to this database after the initial search. Results Temperature Increased temperature has been linked with an increased risk of human WNV cases in several studies, due to the increase in abundance of vector mosquito species (Pecoraro et al. 2007; Roth et al. 2009; Deichmeister and Telang 2010; Morin and Comrie 2010; Reisen et al. 2010). The vector species of WNV generally appear to be able to reproduce and survive for longer periods of time under warmer conditions. One article discusses an increase in temperature in the context of the overwintering ability of vector species (Reisen et al. 2010). In areas such as California and Florida, winter temperatures have the potential to be mild enough to allow for survival of some mosquitoes from the previous year. If these mosquitoes are infected with WNV, it is possible that an increase in WNV cases will be seen the next year due to the increased prevalence of mosquito vectors. Also, it has been identified that range expansion of vector mosquitoes, and consequently human WNV infections, have geographically expanded to areas with increased temperatures (Roth et al. 2009). The expansion of WNV into British Columbia is an example of the relationship between WNV and increased temperature. A large portion of Canada has been infected with WNV since 2002, with British Columbia being the only southern province absent of the infection until 2009. The exact cause of this 7 year gap between infection of the majority of Canada and the infection of British Columbia is unknown; however, in 2009 temperatures were above the 10-­‐year average. Additionally, the area where the first human WNV cases were found was in the hottest part of British Columbia (Roth et al. 2009). Precipitation Precipitation is another variable that is widely discussed as having an effect on human WNV infections; however, a direct causal relationship is unable to be drawn. (Pecoraro et al. 2007; Deichmeister and Telang 2010; Morin and Comrie 2010; Walsh 2012)). Studies that have found low levels of precipitation to be linked to an increase in WNV infections in humans have generally done so in urban areas (Deichmeister and Telang 2010). Urban areas generally have an extensive storm drain system to handle runoff. During periods of high rain this storm drain system consists mostly of moving water, which is an unsuitable habitat for mosquitoes. However, during periods of low rainfall, or drought, water within the system becomes stagnant, and mosquitoes are able to reproduce much more effectively. The large amount of suitable breeding grounds for vector species in urban areas leads to an increase in abundance, and thus, an increase in WNV human infections. Other studies have found precipitation not to be correlated to WNV infection in humans (Pecoraro et al. 2007). Although this study examined some urban and suburban areas, it also included areas outside of the urban environment. It is possible then, that precipitation plays a large role in urban areas and is less important in more rural settings. Also, the amount of water naturally present in an environment may determine the effect rainfall has on mosquito abundance. Since mosquitoes need water to reproduce, in severe drought conditions where no standing water is present, mosquito abundance will be reduced (Morin and Comrie 2010). However, in periods of drought where reproductive areas still exist for mosquitoes (such as storm drain systems in urban areas), abundance may increase due to reproductive habitats being undisturbed by rainfall. Therefore, it may be the case that suitable habitats for reproduction (surface water) are more important than the absolute amount of rainfall received by a region (Walsh 2012). Urbanization Urbanization has been linked to increased abundance of mosquito vector species (Pecoraro et al. 2007; Peterson et al. 2008; Deichmeister and Telang 2010;). In one study, which looked at the abundance and variety of species in rural, suburban, and urban sites, the urban sites had a significantly larger number of common WNV vector species Cx. pipiens (Pecoraro et al. 2007). In addition to the higher abundance of this vector species in urban sites, the variety of species was much lower when compared to rural sites. Rural sites tended to have a much greater diversity of mosquito species when compared to an urban environment, and many of the rural species are not known WNV carriers, possibly reducing the risk of infection outside of urban areas. Therefore, the higher risk of WNV infection in urban areas can be partially attributed to the lower species diversity and higher abundance of vector species. While it is known that urban sites are correlated with increased WNV prevalence, there may be several causes for this increase. One such cause may be the interplay between precipitation, storm drain systems, and the mosquito’s reproductive behaviors (Deichmeister and Telang 2010). Another cause may be the reduction in vegetation present in cities. It has been found that a reduction in the amount of vegetation in an area is correlated with increased WNV transmission (Peterson et al. 2008). This has obvious implications for urban areas where vegetation is nearly absent. The large reduction in vegetation resultant from urbanization may be a factor driving the increase in WNV infection in cities. However, this correlation may also help to explain another infection pattern seen in rural areas of the upper Midwest. In the upper Great Plains region, there is a proportionally higher incidence of WNV infection after normalization by population (Young and Jensen 2012). This area of the United States is mostly used for agriculture and is naturally grassland. Compared to the forested areas of much of the rest of the United States, the relatively low vegetation in this area may help to explain the increased incidence of infections. Hydrogeography Hydrogeography is the study of the orientation and amount of water across a landscape. It has been found that an increase in hydrogeographic area (that is, an increase in the density of surface water present) will result in a decrease of WNV cases in humans (Walsh 2012). While this may seem counterintuitive at first, due to mosquitoes’ requirement for surface water to reproduce, the feeding behavior of mosquitoes lends an explanation. The vector species responsible for transmission of WNV to humans feed on both birds and mammals at various rates, and most species prefer birds as hosts. Since birds require a large amount of water, an increase in hydrogeographic area results in an increase of bird population density. With more birds available for feeding the vector mosquito species, the mosquitoes will feed on birds at a higher rate than they feed on mammals, thus reducing the transmission to humans. Mosquito Feeding Patterns and Birds The vector species mosquito’s diet varies from region to region, and with the abundance of hosts. Generally, vector species feed mainly on birds and mammals, with a preference for birds (Ward et al. 2008; Farajollahi et al. 2011; Molaei et al. 2011) (Figure 4). The way by which many humans become infected with WNV is when they are bitten by a mosquito that has previously fed on a WNV infected bird. Therefore, identifying the prevalence of WNV in bird species, and the feeding patterns of species of vector mosquitoes may allow for prediction and modeling of WNV human infections. Crows have been reported to be a good indicator species for WNV prevalence in a region, since crows are especially sensitive to WNV and carry a high viral load (Eidson et al. 2005; Bouden et al. 2008). Reporting and analyzing data of dead crows and infection rates of crows in an area may be a good way to predict the imminent introduction of WNV into a region (Eidson et al. 2005). The interplay of crows and mosquito populations also affects the prevalence of WNV and modeling can be used to predict epidemics (Bouden et al. 2008). While these studies suggest a simple relationship between crows and WNV infection in humans, the true interaction between vector mosquitoes and their hosts is much more complex. Two species of mosquitoes appear to be prevalent in urban areas throughout the world and act as vectors for WNV, Culex pipiens and Cx. quinquefasciatus (Farajollahi et al. 2011). In addition to these two species, Cx restuans and Cx salinarius have also been identified as vectors in certain areas of the United States (Molaei et al. 2006; Hamer et al. 2008). All of these species have a clear preference for bird hosts compared to mammals; however, crows constitute a very small portion of the mosquito’s diet. By sampling mosquitoes and analyzing their last blood meal, it was found that American robins and sparrows were much more common hosts than crows (Kilpatrick et al. 2006, Molaei et al. 2006). Given this knowledge of feeding patterns of mosquitoes, it seems logical to measure the prevalence of WNV and death of these more common hosts instead of crows. One study investigated the reason for an increase in WNV prevalence during the late summer months (Kilpatrick et al. 2006). Near the end of summer, mosquito’s preferred bird species hosts start to migrate away from the area. In the northeastern portion of the United States, the preferred host for Cx. pipiens is the American robin, which starts to migrate during the later summer months. This decreased availability of its preferred host causes Cx. pipiens to search for new hosts in order to feed. Therefore, the mosquito feeds more on human hosts during this time. Unsurprisingly, this change in feeding pattern leads to a greater infection rate in humans (Kilpatrick et al. 2006). Modeling The use of modeling to predict the number of human cases of WNV can take one of a few different forms including: directly modeling human cases of WNV, modeling the distribution and abundance of mosquito vector species, or modeling the non-­‐human host populations such as birds or horses. The most desirable way to model WNV is to predict directly the number of human cases. If an accurate model can be created to predict the number of human cases of WNV then it simplifies the process and reduces the leaps in logic required to go from either mosquito or non-­‐human host data to human infection. Several attempts have been made to create an accurate model to predict human cases with some success (Peterson et al. 2008; Liu et al. 2009). These studies integrate climate variables (temperature, precipitation, etc), mosquito abundance, and WNV-­‐positive bird abundance into their models in an effort to model the patterns of human infection as closely as possible. One study, while finding some significant results, was limited by a small number of infections and a lack of consistency in mosquito trapping efforts over the study period (Liu et al. 2009). Another study also found limitations in their effort to model human infections in the form of an inability to know exactly where an infection had occurred, compared to where that infected person lived. Also in this model, differences in the distribution of people were not taken into account, so large concentrations of people (such as large urban centers) skewed the data (Peterson et al. 2008). Together these two studies demonstrate the difficulty in creating an effective model for human WNV infections. Other studies have sought to model and describe mosquito or non-­‐human host abundance and infections (Bouden et al. 2008; Ward et al. 2009; Morin and Comrie 2010;). One study sought to model the interactions between mosquitoes and birds, using crows as the primary mosquito host (Bouden et al. 2008). While this study met with some success, its concentration on a small number of variables leaves it vulnerable to unexpected changes. Another study investigated the interdependence of temperature and precipitation and its effect on a specific mosquito species (Morin and Comrie 2010). A final study looked at the effect of a large number of variables on infections of horses with WNV (Ward et al. 2009). Taken together one can see there is a large and highly variable effort under way to model different aspects of the WNV infection cycle. The ultimate goal appears to be modeling human risk and infections; however, this effort is hampered by a large number of variables. In response, efforts to model these variables have given rise to some success, yet leave room for interpretive error as the results of these models must be further applied if human risk and infection is to be determined. Discussion It is clear that WNV will remain prevalent in North America for the foreseeable future. The quick spread of the virus throughout the continent, along with the retention of the virus from year to year in previously infected areas, shows this characteristic. Additionally, the public health concerns brought about by this virus ensures it will remain a high priority. The current research has identified certain mosquito species which are vital to the transmission of the disease and non-­‐human hosts which serve as a reservoir for the virus. Therefore, study and control of the transmission agents and reservoirs are imperative to controlling human cases of WNV. Thus far, increased temperature has nearly uniformly been identified as a risk factor for increased WNV cases in humans. This has to do with an increase in mosquito breeding and feeding frequency in periods of hot weather. Precipitation seems to affect mosquito distribution and abundance; however, controversy remains regarding what extent this variable plays in WNV infections in humans. It seems likely that precipitation alters infection rates in humans by affecting reproductive habitats of mosquitoes. Therefore regional hydrogeography, climate, and other landscape variables (cities, mountains, etc.) play a role in determining the significance of precipitation on vector species populations, and thus, human WNV infections. Urbanization appears to result in an increased risk of human infection. This stems from the interplay between rainfall, storm drains, and reproductive habitats for vector species. Cities also tend to support a smaller variety of mosquito species, so vector species play a more prominent role. Many of the vector species feed on birds as their primary source of food; however, the crow has been singled out as an indicator species. While vector species do feed on the crow, it is a very small portion of the mosquito’s diet, and therefore, other birds may provide a more accurate description of the disease dynamics of WNV within a community. For many studies, modeling and prediction is the goal of testing and manipulating variables affecting mosquito population. Humans care about WNV, as with any disease, because of the health implications on humans. The ability to accurately model and predict infection rates in space and time will lead to more effective treatment, and perhaps more importantly, better control of the disease through manipulation of the mosquito population. In the future, climate change will continue to affect many of the variables that determine the distribution and abundance of vector mosquito species. Temperature is increasing, and variation in precipitation amount is becoming more common. This has implications for mosquitoes in the form of overwintering ability and range expansion (Hongoh et al. 2012; Roth et al. 2010; Reisen et al. 2010). With increasing temperatures vector species such as Culex pipiens may be able to survive in habitats farther north into Canada, thus exposing a larger number of people to WNV (Hongoh et al. 2012; Roth et al. 2010). In addition, increasing temperatures in more mild climates will allow a greater proportion of the mosquitoes to successfully over winter, possibly increasing the abundance of mosquitoes for the next year (Reisen et al. 2010). Moreover, the increased temperatures throughout the current habitats of vector species during breeding season may increase their abundance and biting frequency significantly. In conclusion, WNV is a serious vector borne illness that is only in the beginning stages of transmission in the Western Hemisphere. While some variables have shown a consistent predictive pattern, others variables and their link with WNV remain unclear and further study is warranted. Furthermore, the interaction between variables must be investigated, and effective models need to be produced in order to most effectively control transmission to humans. Literature Cited Andre, F.E., R. Booy, H.L. Bock, J. Clemens, S.K. Datta, T.J. John, B.W. Lee, S. Lolekha, H. Peltola, T.A. Ruff, M. Santosham, and H.J. Schmitt. 2008. Vaccination greatly reduces disease, disability, death, and inequity worldwide. Bulletin of the World Health Organization. 86(2): 140-­‐146. Begon, M., C.R. Townsend, and J.L. Harper. 2006. Parasitism and disease. Pages 347-­‐380 in Ecology: From individuals to ecosystems. Blackwell Publishing Ltd., Oxford, UK. Bouden, M., B. Moulin, and P. Gosselin. 2008. The geosimulation of West Nile virus propagation: a multi-­‐agent and climate sensitive tool for risk management in public health. International Journal of Health Geographics. 7: 35-­‐54. Deichmeister, J.M., and A. Telang. 2011. Abundance of West Nile virus mosquito vectors in relation to climate and landscape variables. Journal of Vector Ecology. 36(1): 75-­‐85. Eidson, M., K. Schmit, Y. Hagiwara, M. Anand, P.B. Backenson, I. Gotham, and L. Kramer. 2005. Dead crow density and West Nile virus monitoring, New York. Emerging Infectious Diseases. 11(9): 1370-­‐1375. Goldblum, N., W. Jasinska-­‐Klingberg, M.A. Klingberg, and V.V. Sterk. 1956. The natural history of West Nile fever. I. Clinical observations during an epidemic in Israel. American Journal of Hygiene. 64(3): 259-­‐269. Farajollahi, A., D.M. Fonseca, L.D. Kramer, and A.M. Kilpatrick. 2011. "Bird biting" mosquitoes and human disease: a review of the role of Culex pipiens complex mosquitoes in epidemiology. Infection, Genetics, and Evolution. 11: 1577-­‐1585. Hamer, G.L., U.D. Kitron, J.D. Brawn, S.R. Loss, M.O. Ruiz, T.L. Goldberg, and E.D. Walker. 2008. Culex pipiens (Diptera: Culicidae): A bridge vector of West Nile virus in humans. Journal of Medical Entomology. 45(1): 125-­‐128. Hongoh, V., L. Berrang-­‐Ford, M.E. Scott, and L.R. Lindsay. 2012. Expanding geographical distribution of the mosquito Culex pipiens, in Canada under climate change. Applied Geography. 33: 53-­‐62. Kilpatrick, A.M., L.D. Kramer, M.J. Jones, P.P. Marra, and P. Daszak. 2006. West Nile virus epidemics in North America are driven by shifts in mosquito feeding behavior. PLoS Biology. 4(4): e82. Lanciotti, R.S., J.T. Roehrig, V. Deubel, and J. Smith et al. 1999. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science. 286: 2333-­‐2337. Lui, A., V. Lee, D. Galusha, M.D. Slade, M. Diuk-­‐Wasser, T. Andreadis, M. Scotch, and P.M. Rabinowitz. 2009. Risk factors for human infection with West Nile virus in Connecticut: a multi-­‐year analysis. International Journal of Health Geographics. 8: 67-­‐77. Molaei, G., T.G. Andreadis, P.M. Armstrong, J.F. Anderson, and C.R. Vossbrinck. 2006. Host feeding patterns of the Culex mosquitoes and the West Nile virus transmission, northeastern United States. Emerging Infectious Diseases. 12(3): 468-­‐474. Morin, C.W., and A. C. Comrie. 2010. Modeled response of the west nile virus vector Culex quinquefasciatus to changing climate using the dynamic mosquito simulation model. International Journal of Biometeorology. 54: 517-­‐529. Pecoraro, H.L., H.L. Day, R. Reineke, N. Stevens, J.C. Withey, J.M. Marzluff, and J.S. Meschke. 2007. Climate and landscape correlates for potential West Nile virus mosquito vectors in the Seattle region. Journal of Vector Ecology. 32(1): 22-­‐28. Peterson, A.T., A. Robbins, R. Restifo, J. Howell, and R. Nasci. 2008. Predictable ecology and geography of West Nile virus transmission in the central United States. Journal of Vector Ecology. 33(2): 342-­‐352. Reisen, W.K., T. Thiemann., C.M. Barker, H. Lu, B. Carroll, Y. Fang, and H.D. Lothrop. 2010. Effects of warm winter temperature on the abundance and gonotronphic activity of Culex (diptera: culicidae) in California. Journal of Medical Entomology. 47(2): 230-­‐237. Roth, D., B. Henry, S. Mak, M. Fraser, M. Taylor, M. Li, K. Cooper, A. Furnell, Q. Wong, and M. Morshed. 2010. West Nile range expansion into british columbia. Emerging Infectious Diseases. 16(8): 1251-­‐1258. Smithburn, K.C., T.P. Hughes, A.W. Burke, and J.H. Paul. 1940. A neurotropic virus isolated from the blood of a native of Uganda. American Journal of Tropical Medicine. S1-­‐20(4): 471-­‐492. Tsai, T.F., F. Popovici, C. Cernescu, G.L. Campbell, and N.I. Nedelcu. 1998. West Nile encephalitis epidemic in southeastern Romania. The Lancet. 352: 767-­‐771. Walsh, M.G. 2012. The role of hydrogeography and climate in the landscape epidemiology of west nile virus in New York State from 2000 to 2010. PLoS One. 7(2): e30620. Ward, M.P., C.A. Wittich, G. Fosgate, and R. Srinivasan. 2008. Environmental risk factors for eqine West Nile virus disease cases in Texas. Veterinary Research Communications. 33: 461-­‐471. Young, S.G., and R.R. Jensen. 2012. Statistical and visual analysis of human West Nile virus infection in the United States, 1999-­‐2008. Applied Geography. 34: 425-­‐431. Figure 1: This figure shows the genetic relationships between different strains of WNV based on a the nucleotide sequence of a specific protein. It has been determined that the first WNV infection in humans in New York was most closely related to a strain isolated in Israel during the previous year (Lanciotti et al. 1999). Figure 2: Shown here is the absolute number of WNV cases throughout the country from introduction in 1999 to 2008. Each dot is equal to 5 cases. Notice the clustering of cases near urban centers, the Mississippi River Valley, and throughout the upper Mid-­‐West (Young and Jensen 2012). Figure 3: This proportional map of human WNV infections between 1999-­‐2008 demonstrates the significance of the clustering of cases near urban centers and the upper mid-­‐west region. It appears that while urbanization plays some role in increasing human WNV infections, other variables are at work in some regions of the country (Young and Jensen 2012). Figure 4: The proportions of hosts in 4 vector species diets. The left vertical axis shows proportion for bird and mammal feedings, while the right vertical axis shows proportions for human feedings. All 4 vector species show a significant preference for birds when compared to mammals, with humans making up a small percentage of mammal feedings (Farajollahi et al 2011).