Continental-scale dynamics of avian influenza in U.S. waterfowl are driven by demography, migration, and temperature.

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.

Fluid Spatial Dynamics of West Nile Virus in the United States: Rapid Spread in a Permissive Host Environment.

UNLABELLED: 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.

Letter to the Editor: Venezuelan Equine Encephalitis virus 1B Invasion and Epidemic Control-South Texas, 1971.

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.

Influenza infection in wild raccoons.

Raccoons (Procyon lotor) are common, widely distributed animals that frequently come into contact with wild waterfowl, agricultural operations, and humans. Serosurveys showed that raccoons are exposed to avian influenza virus. We found antibodies to a variety of influenza virus subtypes (H10N7, H4N6, H4N2, H3, and H1) with wide geographic variation in seroprevalence. Experimental infection studies showed that raccoons become infected with avian and human influenza A viruses, shed and transmit virus to virus-free animals, and seroconvert. Analyses of cellular receptors showed that raccoons have avian and human type receptors with a similar distribution as found in human respiratory tracts. The potential exists for co-infection of multiple subtypes of influenza virus with genetic reassortment and creation of novel strains of influenza virus. Experimental and field data indicate that raccoons may play an important role in influenza disease ecology and pose risks to agriculture and human health.

Potential effect of prior raccoonpox virus infection in raccoons on vaccinia-based rabies immunization.

BACKGROUND: The USDA, Wildlife Services cooperative oral rabies vaccination (ORV) program uses a live vaccinia virus-vectored (genus Orthopoxvirus) vaccine, Raboral V-RG (V-RG), to vaccinate specific wildlife species against rabies virus in several regions of the U.S. Several naturally occurring orthopoxviruses have been found in North America, including one isolated from asymptomatic raccoons (Procyon lotor). The effect of naturally occurring antibodies to orthopoxviruses on successful V-RG vaccination in raccoons is the focus of this study. RESULTS: Overall, raccoons pre-immunized (n = 10) with a recombinant raccoonpox virus vaccine (RCN-F1) responded to vaccination with V-RG with lower rabies virus neutralizing antibody (VNA) titers than those which were not pre-immunized (n = 10) and some failed to seroconvert for rabies VNA to detectable levels. CONCLUSION: These results suggest that the success of some ORV campaigns may be hindered where raccoonpox virus or possibly other orthopoxvirus antibodies are common in wildlife species targeted for ORV. If these areas are identified, different vaccination strategies may be warranted.

Influenza exposure in United States feral swine populations.

Swine play an important role in the disease ecology of influenza. Having cellular receptors in common with birds and humans, swine provide opportunities for mixed infections and potential for genetic reassortment between avian, human, and porcine influenza. Feral swine populations are rapidly expanding in both numbers and range and are increasingly coming into contact with waterfowl, humans, and agricultural operations. In this study, over 875 feral swine were sampled from six states across the United States for serologic evidence of exposure to influenza. In Oklahoma, Florida, and Missouri, USA, no seropositive feral swine were detected. Seropositive swine were detected in California, Mississippi, and Texas, USA. Antibody prevalences in these states were 1% in Mississippi, 5% in California, and 14.4% in Texas. All seropositive swine were exposed to H3N2 subtype, the predominant subtype currently circulating in domestic swine. The only exceptions were in San Saba County, Texas, where of the 15 seropositive samples, four were positive for H1N1 and seven for both H1N1 and H3N2. In Texas, there was large geographical and temporal variation in antibody prevalence and no obvious connection to domestic swine operations. No evidence of exposure to avian influenza in feral swine was uncovered. From these results it is apparent that influenza in feral swine poses a risk primarily to swine production operations. However, because feral swine share habitat with waterfowl, prey on and scavenge dead and dying birds, are highly mobile, and are increasingly coming into contact with humans, the potential for these animals to become infected with avian or human influenza in addition to swine influenza is a distinct possibility.

Fox squirrel (Sciurus niger) associations with West Nile virus.

Tree squirrels (Sciurus spp.) have been recently shown to be commonly exposed to West Nile virus (WNV). Many characteristics of WNV infections in tree squirrels are unknown. To better understand WNV associations in fox squirrels (S. niger), we conducted mark-recapture sampling (N = 72) and radio telemetry to study the longitudinal seroprevalence, seroconversions, and ectoparasites of these animals during 2005-2006 in northern Colorado. Five seroconversions were documented during this study. The majority of seroconversions occurred during the late summer/fall months. However, one seroconversion was documented over the time period of February to late March 2005. Fleas (Orchopeas howardi) were tested for WNV RNA using real-time PCR techniques. No WNV RNA positive fleas (N = 33) were detected. In addition, urine samples (N = 17) opportunistically collected from fox squirrels were negative for WNV RNA. Results indicate that seroconversions can be observed in fox squirrels during low WNV transmission years.

Experimental infection of fox squirrels (Sciurus niger) with West Nile virus.

Tree squirrels (Sciurus spp.) have exhibited high seroprevalence rates, suggesting that they are commonly exposed to West Nile virus (WNV). Many characteristics of WNV infections in tree squirrels, such as the durations and levels of viremia, remain unknown. To better understand WNV infections in fox squirrels (S. niger), we subcutaneously inoculated fourteen fox squirrels with WNV. Peak viremias ranged from 10(4.00) plaque-forming units (PFU)/mL of serum on day 2 post-infection (DPI) to 10(4.98) PFU/mL on 3 DPI, although viremias varied between individuals. Oral secretions of some fox squirrels were positive for WNV viral RNA, occasionally to moderate levels (10(3.2) PFU equivalent/swab). WNV PFU equivalents in organs were low or undetectable on 12 DPI; gross and histologic lesions were rare. The viremia profiles of fox squirrels indicate that they could serve as amplifying hosts in nature. In addition, viral RNA in the oral cavity and feces indicate that this species could contribute to alternative WNV transmission in suburban communities.

Serologic evidence of exposure of wild mammals to flaviviruses in the central and eastern United States.

Serosurveys were conducted to obtain flavivirus and West Nile virus (WNV) seroprevalence data from mammals. Sera from 513 small- and medium-sized mammals collected during late summer and fall 2003 from Colorado, Louisiana, New York, Ohio, and Pennsylvania were screened for flavivirus-specific antibodies. Sera samples containing antibody to flaviviruses were screened for WNV-specific antibodies by epitope-blocking enzyme-linked immunosorbent assays and confirmed with plaque reduction neutralization tests. Prevalence of WNV antibodies among study sites ranged from 0% to 42.8% among the mammal communities sampled. High prevalence rates for WNV were noted among raccoons (100%, with a very small sample size, N = 2), Virginia opossums (50.0%), fox squirrels (49.1%), and eastern gray squirrels (48.3%). The high WNV antibody prevalence noted for tree squirrels, the peri-domestic tendencies of several of these species, and their ease of observation could make these species useful sentinels for monitoring WNV activity within urban communities.

WEST NILE VIRUS ANTIBODY DECAY RATE IN FREE-RANGING BIRDS.

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.

West Nile virus infection in American Robins: new insights on dose response.

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.

Graphically characterizing the movement of a rabid striped skunk epizootic across the landscape in northwestern Wyoming.

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 km(2) 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.

Surveillance of Wildlife Diseases: Lessons from the West Nile Virus Outbreak.

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?

Raccoon (Procyon lotor) movements and dispersal associated with ridges and valleys of Pennsylvania: implications for rabies management.

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.

Antibody responses of raccoons naturally exposed to influenza A virus.

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.

Prevalence of West Nile virus in migratory birds during spring and fall migration.

To investigate the role of migratory birds in the dissemination of West Nile virus (WNV), we measured the prevalence of infectious WNV and specific WNV neutralizing antibodies in birds, principally Passeriformes, during spring and fall migrations in the Atlantic and Mississippi flyways from 2001-2003. Blood samples were obtained from 13,403 birds, representing 133 species. Specific WNV neutralizing antibody was detected in 254 resident and migratory birds, representing 39 species, and was most commonly detected in northern cardinals (Cardinalis cardinalis) (9.8%, N = 762) and gray catbirds (Dumetella carolinensis) (3.2%, N = 3188). West Nile virus viremias were detected in 19 birds, including 8 gray catbirds, and only during the fall migratory period. These results provide additional evidence that migratory birds may have been a principal agent for the spread of WNV in North America and provide data on the occurrence of WNV in a variety of bird species.

Experimental infection of cliff swallows (Petrochelidon pyrrhonota) with varying doses of West Nile virus.

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 10(6.3) 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.

Ecological correlates of risk and incidence of West Nile virus in the United States.

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.