Emerging diseases of wildlife origin are increasingly spilling over into humans and domestic animals. Surveillance and risk assessments for transmission between these populations are informed by a mechanistic understanding of the pathogens in wildlife reservoirs. For avian influenza viruses (AIV), much observational and experimental work in wildlife has been conducted at local scales, yet fully understanding their spread and distribution requires assessing the mechanisms acting at both local, (e.g., intrinsic epidemic dynamics), and continental scales, (e.g., long-distance migration). Here, we combined a large, continental-scale data set on low pathogenic, Type A AIV in the United States with a novel network-based application of bird banding/recovery data to investigate the migration-based drivers of AIV and their relative importance compared to well-characterized local drivers (e.g., demography, environmental persistence). We compared among regression models reflecting hypothesized ecological processes and evaluated their ability to predict AIV in space and time using within and out-of-sample validation. We found that predictors of AIV were associated with multiple mechanisms at local and continental scales. Hypotheses characterizing local epidemic dynamics were strongly supported, with age, the age-specific aggregation of migratory birds in an area and temperature being the best predictors of infection. Hypotheses defining larger, network-based features of the migration processes, such as clustering or between-cluster mixing explained less variation but were also supported. Therefore, our results support a role for local processes in driving the continental distribution of AIV.

The epidemic strain of Venezuelan equine encephalitis virus (VEE) 1B invaded south Texas in 1971. The success of the eventual containment and control of the virus invasion was the early recognition and immediate detection, cooperation, coordination, and participation among multiple federal agencies. There were 4739 wild vertebrate animals trapped on a ranch in the area with only 1 VEE virus isolation from a Virgina opossum (Didelphis virginiana). A large number of mosquitoes were also collected on the ranch and tested, resulting in 240 VEE virus isolations. Virus isolations were obtained from 58% of the 33 equines tested. Wild vertebrates did not play a significant role in the outbreak.

The raccoon (Procyon lotor) continues to be a prominent terrestrial rabies reservoir in the eastern United States. Describing the dispersal and movements of these animals and determining geographic features that are natural hindrances or corridors to movements could be used to assist oral rabies vaccination efforts. The landscape of the ridge-and-valley system in Pennsylvania exhibits characteristics of both natural potential hindrances and travel corridors to the movements of wildlife. The movements of 49 raccoons were monitored throughout a ridge and two adjacent valley sites to assess their movements related to these landscape features. Results suggest that few raccoons cross the ridge we studied over the short-term and that long-distance movements of these animals are uncommon in this region. Consequently, movement corridors within the ridge were largely confined to spur valleys within the ridge system. These results may be useful in strategic oral rabies vaccination programs in Pennsylvania and other areas where natural hindrances and travel corridors to movement are identified.

Sera from white-tailed deer (Odocoileus virginianus) were collected in Iowa during the winter months (1999-2003), 2 years before and after West Nile virus (WNV) was first reported in Iowa (2001), and were analyzed for antibodies t o WNV. Samples from 1999 to 2001 were antibody negalive by blocking en~yrr~e-l i~iked ~ I ~ I I I ~ U I I U ~ U I L I ~ I I ~ assay (bELISA) and plaque reduction neutralization test (PRNT,,). Prevalence derived from bELISA (2002, 12.7%; 2003. 11.2%) and WNV PRNT,, (2002.7.9%; 2003, 8.5%) assays were similar. All sanlples were negative for antibodies against St. Louis encephalitis virus as determined by PRNT,,,. Antibodies to flaviviruses were detected by indirect enzyme-linked immunosorbent assay (iELISA) prior to the first WNV cases reported in Iowa (1999-2001) with prevalence ranging from 2.2% to 3.2%, suggesting the circulation of an additional undescribed flavivirus prior to the introduction of WNV into the area. Flavivirus prevalence as determined by iELISA increased in 2002 and 2003 (23.3% and 31.9%, respectively). The increase in prevalence exceeded estimates of WNV prevalence. suggesting that conditions favored general flavivirus transmission (including WNV) during the 2002-2003 epizootic. These data indicate that serologic analysis of deer sera collected from hunter harvests may prove useful for surveillance and evidence of local transmission of WNV and other pathogens and identify white-tailed deer as a species for further studies for host competency.

Antibody duration, following a humoral immune response to West Nile virus (WNV) infection, is poorly understood in free-ranging avian hosts. Quantifying antibody decay rate is important for interpreting serologic results and for understanding the potential for birds to serorevert and become susceptible again. We sampled free-ranging birds in Chicago, Illinois, US, from 2005 to 2011 and Atlanta, Georgia, US, from 2010 to 2012 to examine the dynamics of antibody decay following natural WNV infection. Using serial dilutions in a blocking enzyme-linked immunosorbent assay, we quantified WNV antibody titer in repeated blood samples from individual birds over time. We quantified a rate of antibody decay for 23 Northern Cardinals (Cardinalis cardinalis) of 0.198 natural log units per month and 24 individuals of other bird species of 0.178 natural log units per month. Our results suggest that juveniles had a higher rate of antibody decay than adults, which is consistent with nonlinear antibody decay at different times postexposure. Overall, most birds had undetectable titers 2 yr postexposure. Nonuniform WNV antibody decay rates in free-ranging birds underscore the need for cautious interpretation of avian serology results in the context of arbovirus surveillance and epidemiology.

ABSTRACT The introduction of West Nile virus (WNV) into North America in 1999 is a classic example of viral emergence in a new environment, with its subsequent dispersion across the continent having a major impact on local bird populations. Despite the importance of this epizootic, the pattern, dynamics, and determinants of WNV spread in its natural hosts remain uncertain. In particular, it is unclear whether the virus encountered major barriers to transmission, or spread in an unconstrained manner, and if specific viral lineages were favored over others indicative of intrinsic differences in fitness. To address these key questions in WNV evolution and ecology, we sequenced the complete genomes of approximately 300 avian isolates sampled across the United States between 2001 and 2012. Phylogenetic analysis revealed a relatively star-like tree structure, indicative of explosive viral spread in the United States, although with some replacement of viral genotypes through time. These data are striking in that viral sequences exhibit relatively limited clustering according to geographic region, particularly for those viruses sampled from birds, and no strong phylogenetic association with well-sampled avian species. The genome sequence data analyzed here also contain relatively little evidence for adaptive evolution, particularly of structural proteins, suggesting that most viral lineages are of similar fitness and that WNV is well adapted to the ecology of mosquito vectors and diverse avian hosts in the United States. In sum, the molecular evolution of WNV in North America depicts a largely unfettered expansion within a permissive host and geographic population with little evidence of major adaptive barriers. IMPORTANCE How viruses spread in new host and geographic environments is central to understanding the emergence and evolution of novel infectious diseases and for predicting their likely impact. The emergence of the vector-borne West Nile virus (WNV) in North America in 1999 represents a classic example of this process. Using approximately 300 new viral genomes sampled from wild birds, we show that WNV experienced an explosive spread with little geographical or host constraints within birds and relatively low levels of adaptive evolution. From its introduction into the state of New York, WNV spread across the United States, reaching California and Florida within 4 years, a migration that is clearly reflected in our genomic sequence data, and with a general absence of distinct geographical clusters of bird viruses. However, some geographically distinct viral lineages were found to circulate in mosquitoes, likely reflecting their limited long-distance movement compared to avian species.

West Nile virus (WNV) is a vector-borne pathogen that was first detected in the United States in 1999. The natural transmission cycle of WNV involves mosquito vectors and avian hosts, which vary in their competency to transmit the virus. American robins are an abundant backyard species in the United States and appear to have an important role in the amplification and dissemination of WNV. In this study we examine the response of American robins to infection with various WNV doses within the range of those administered by some natural mosquito vectors. Thirty American robins were assigned a WNV dosage treatment and needle inoculated with 10(0.95) PFU, 10(1.26) PFU, 10(2.15) PFU, or 10(3.15) PFU. Serum samples were tested for the presence of infectious WNV and/or antibodies, while oral swabs were tested for the presence of WNV RNA. Five of the 30 (17%) robins had neutralizing antibodies to WNV prior to the experiment and none developed viremia or shed WNV RNA. The proportion of WNV-seronegative birds that became viremic after WNV inoculation increased in a dose dependent manner. At the lowest dose, only 40% (2/5) of the inoculated birds developed productive infections while at the highest dose, 100% (7/7) of the birds became viremic. Oral shedding of WNV RNA followed a similar trend where robins inoculated with the lower two doses were less likely to shed viral RNA (25%) than robins inoculated with one of the higher doses (92%). Viremia titers and morbidity did not increase in a dose dependent manner; only two birds succumbed to infection and, interestingly, both were inoculated with the lowest dose of WNV. It is clear that the disease ecology of WNV is a complex interplay of hosts, vectors, and viral dose delivered.

A striped skunk (Mephitis mephitis) rabies epizootic in northwestern Wyoming was studied from the Index Case in 1988 to the last case in 1993, and possibly is the first rabies epizootic in a previously rabies-free zone monitored from beginning to end. The 843 km2 study area comprised skunk habitat along 90 km of Shoshone River’s floodplain from Bighorn Lake upstream to Cody. Of 1,015 skunks tested, 215 were rabies-positive. Integrating spatial and temporal data from positive cases, we analyzed the epizootic’s movements and dynamics at 6-month intervals using multivariate movement maps, a new multivariate descriptive methodology presented here to demonstrate the epizootic’s directional flow, while illustrating areas with higher case densities (i.e., wave crests). This approach should help epidemiologists and public health officials to better understand future rabies epizootics.

The West Nile virus outbreak of 1999 revealed many weaknesses in this country's ability to respond to disease threats that cross species lines. There were issues of poor communication among human, domestic animal, and wildlife health agencies that delayed diagnosis; a lack of diagnostic capacity of wildlife agencies at the state level; the exclusion of captive wildlife from any surveillance efforts; an inability to visualize the geospatial relationship between the human and avian outbreaks in a timely manner; and marked disparities of funding levels across agencies. Wildlife has played an important role in recent emerging infectious diseases, and it is clear that a One Health approach will be necessary to respond to future threats. The question is, are we any better prepared to recognize and respond to a wildlife-related emerging infectious disease than we were 14 years ago? Have the lessons of WNV been learned?

Sera from 360 ciconiform and pelecaniform birds collected in Florida (USA) from 1974 to 1990 were tested for serum neutralizing (SN) antibodies to eastern equine encephalitis (EEE), St. Louis encephalitis (SLE), and Everglades (EVE) viruses. Serum neutralizing antibodies to EEE virus were detected in 2%, to SLE virus in 7%, and to EVE virus in none of the samples. Pelecaniform birds (16%) had a higher antibody prevalence (P < 0.02) for SLE virus than did ciconiform birds (5%). Virus could not be isolated from 67 samples. Nestling birds with SN antibodies to both EEE and SLE viruses were found in both fresh water and marine colonies. Antibodies were more prevalent in adult and fledged juvenile birds than in nestlings.

Studying the lower Shoshone River Basin's (SRB) striped skunk (Mephitis mephitis) rabies 6-year epizootic in northwestern Wyoming has produced four didactic lessons. First, physiographic changes by settlers circa 1900 affected its zoogeography by creating a canal system for irrigating crops originating at Buffalo Bill Reservoir (BBR). The resulting landscape changes increased agricultural lands and skunk habitat eightfold between the valley's steep gravel benches. The valley was historically free of skunk rabies until the epizootic's index case in August 1988. Second, human intervention began when the Bighorn County Predator Board (BCPB) proactively implemented rabies trapping surveillance and depopulation programs in 1989. The epizootic's second case occurred in February 1989. From 1990-1993, the U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife Services (WS) Wyoming office continued these programs. The epizootic ended in 1993 with over 200 rabid animals diagnosed by the Wyoming State Veterinary Laboratory (WSVL) from about 1,000 skunks tested. Cooperator agreements began in January 1989 and continued throughout the epizootic for trapping and shooting of potential vectors. Third, WS National Wildlife Research Center (NWRC) analyzed the epizootic movements using monthly mean movements and standardized ellipsoids. These analyses demonstrated that rabies dispersed radially from the index case until it reached the Shoshone River. From there, the epizootic spread downstream until stopped by Bighorn Lake (BL) in 1989 and upstream until stopped by BBR in 1992. Fourth, descriptive multivariate movement maps (MMMs) of the epizootic were analogous to fluid wave characteristics describing a swell moving along the surface of a liquid as the "leading edge" and "crest." All rabid animal locations were derived from the first use of global positioning system (GPS) in investigating wildlife disease. These lessons learned should assist others to better understand skunk rabies epizootics.

The raccoon (Procyon lotor) continues to be a prominent terrestrial rabies reservoir in the eastern United States. Describing the dispersal and movements of these animals and determining geographic features that are natural hindrances or corridors to movements could be used to assist oral rabies vaccination efforts. The landscape of the ridge-and-valley system in Pennsylvania exhibits characteristics of both natural potential hindrances and travel corridors to the movements of wildlife. The movements of 49 raccoons were monitored throughout a ridge and two adjacent valley sites to assess their movements related to these landscape features. Results suggest that few raccoons cross the ridge we studied over the short-term and that long-distance movements of these animals are uncommon in this region. Consequently, movement corridors within the ridge were largely confined to spur valleys within the ridge system. These results may be useful in strategic oral rabies vaccination programs in Pennsylvania and other areas where natural hindrances and travel corridors to movement are identified.

The cliff swallow (Petrochelidon pyrrhonota, Vieillot) could potentially play an important role in the transmission of West Nile virus (WNV) because of its breeding ecology, reservoir competence status, and potentially high natural exposure rates. These birds nest in colonies and their nests are occupied year round by swallow bugs (Oeciacus vicarius, Hovarth), hematophagus ecto-parasites that feed primarily on cliff swallows. Swallow bugs are most likely exposed to WNV while feeding on infectious blood of cliff swallows and, thus, if competent vectors, could contribute to initiation and maintenance of seasonal WNV transmission. In addition, swallow bugs remain within nests year round and, therefore, if persistently infected and competent vectors, they could provide an overwintering mechanism for WNV. We tested the hypothesis that swallow bugs become infected with WNV through direct abdominal inoculation or ingestion of infectious blood meals. We observed that swallow bugs did not maintain or amplify WNV, and infectious virus titers within bugs declined over 15 d postexposure. These results suggest that swallow bugs may not be competent vectors of WNV, and therefore are unlikely to play a significant role in transmission.

The cliff swallow (Petrochelidon pyrrhonota) could play an important role in the transmission of West Nile virus (WNV) because of its breeding ecology, reservoir competence status, and potentially high natural exposure rates. Cliff swallows nest within colonies and their nests are occupied year-round by swallow bugs (Oeciacus vicarius), hematophagus ectoparasites that feed primarily on cliff swallows. These parasites are likely exposed to WNV while feeding on infectious blood of nesting cliff swallow adults and nestlings and thus, if competent vectors, could contribute to seasonal elevations in WNV transmission. In addition, swallow bugs remain within nests year-round and therefore could provide a potential overwintering mechanism for WNV if persistently infected. To test the hypotheses that swallow bugs are competent vectors and become persistently infected with WNV, we experimentally inoculated cliff swallow nestlings, allowed swallow bugs to feed on these birds during the acute phase of infection, and then exposed naive cliff swallow nestlings to the same swallow bugs. In addition, a subset of swallow bugs that fed on infectious swallow nestlings was maintained through a simulated overwintering period. Although swallow bugs ingested infectious blood (up to 10(6.8) plaque-forming units of WNV/mL serum) and subsequently blood-fed on naive swallows, no WNV transmission was detected, and all bugs tested WNV negative after the simulated overwintering period. Although many ecologic scenarios exist beyond the present study, our results suggest that swallow bugs may be unlikely to serve as competent biological vectors for WNV during active transmission periods or to reinitiate seasonal transmission.

To the Editor: A scientifi c debate with public health implications wages: What caused the medieval European plague epidemics known as Black Death? Recent articles note inconsistencies between a rat fl ea– borne pandemic of Yersinia pestis (the bacterium that causes bubonic plague) and the documented characteristics of Black Death (1, among others). Ayyadurai et al. (2) acknowledge that a rat fl ea–only hypothesis does not fi t Black Death observations, but they resolve theoretical transmission inconsistencies through a louse-borne hypothesis. Ayyadurai et al. base their surety of fact—that medieval “plagues” were caused by Y. pestis infection—on a 2007 study (3) in which 5 of 36 teeth of “plague” victims, none of which were dated to the Black Death era (1347–1351), contained biological evidence of Y. pestis. The 3 locations in that study were all port cities: 2 on the Mediterranean Sea and 1 on the Rhone River. As Duncan and Scott (4) note, bubonic plague most likely existed endemically near ship-borne trade, unlike the fast-moving epidemic fronts exhibited by medieval “plagues.” Moreover, Gilbert et al. (5) found no Y. pestis DNA in 61 skeletons from primarily nonport locations in England, France, and Denmark. We do not dispute the authors’ claim that Y. pestis might have been present in some skeletons from port cities in France, or that body lice might, under certain circumstances, transmit the Orientalis biotype of Y. pestis; their work appears careful and considered. However, given the differences mentioned above and improved knowledge on the rapidity of virus mutation and worldwide transmission potential, we merely argue that the simplest explanation for medieval plagues has yet to be ruled out: that they may have resulted from a human-to-human transmitted virus. Adding complexity to an already complicated etiologic theory, and stating such as historical fact based on limited geography and sample size, does not seem congruent with Occam’s razor.

An investigation was performed to describe the responses of naturally acquired antibodies to influenza A virus in raccoons (Procyon lotor) over time. Seven wild raccoons, some of which had been exposed to multiple subtypes of influenza A virus, were held in captivity for 279 days, and serum samples were collected on 10 occasions during this interval. Serum samples from 9 of 10 bleeding occasions were tested using an epitope-blocking enzyme-linked immunosorbent assay for the presence of antibodies to influenza A virus. Although titer declines were noted in most animals over time, all animals maintained detectable antibodies for the duration of the study. These data indicate that naturally acquired antibodies to influenza A virus can remain detectable in raccoons for many months, with the actual duration presumably being much longer because all animals had been exposed to influenza A virus before this study commenced. This information is important to surveillance programs because the duration of naturally acquired antibodies to influenza A virus in wildlife populations is largely unknown.

West Nile virus, which was recently introduced to North America, is a mosquito-borne pathogen that infects a wide range of vertebrate hosts, including humans. Several species of birds appear to be the primary reservoir hosts, whereas other bird species, as well as other vertebrate species, can be infected but are less competent reservoirs. One hypothesis regarding the transmission dynamics of West Nile virus suggests that high bird diversity reduces West Nile virus transmission because mosquito blood-meals are distributed across a wide range of bird species, many of which have low reservoir competence. One mechanism by which this hypothesis can operate is that high-diversity bird communities might have lower community-competence, defined as the sum of the product of each species’ abundance and its reservoir competence index value. Additional hypotheses posit that West Nile virus transmission will be reduced when either: (1) abundance of mosquito vectors is low; or (2) human population density is low. We assessed these hypotheses at two spatial scales: a regional scale near Saint Louis, MO, and a national scale (continental USA). We found that prevalence of West Nile virus infection in mosquito vectors and in humans increased with decreasing bird diversity and with increasing reservoir competence of the bird community. Our results suggest that conservation of avian diversity might help ameliorate the current West Nile virus epidemic in the USA

Cliff swallows (Petrochelidon pyrrhonota) were inoculated with differing doses of West Nile virus (WNV) to evaluate their potential role as reservoir hosts in nature. Swallows often nest in large colonies in habitats and months associated with high mosquito abundance and early WNV transmission in North America. Additionally, cliff swallow diet consists of insects, including mosquitoes, leading to an additional potential route of WNV infection. The average peak viremia titer among infected cliff swallows was 106.3, plaque-forming units (PFU)/mL serum and the reservoir competence index was 0.34. There was no correlation between dose and probability of becoming infected or viremia peak and duration. Oral shedding was detected from 2 to 14 days post-inoculation with an average peak titer of 10(4.4) PFU/swab. These results suggest that cliff swallows are competent reservoir hosts of WNV and therefore, they may play a role in early seasonal amplification and maintenance of WNV.