WNV and SLEV coinfection in avian and mosquito hosts: impact on viremia, antibody responses, and vector competence.

West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related flaviviruses that can cause encephalitis in humans and related diseases in animals. In nature, both are transmitted by Culex, with wild birds, including jays, sparrows, and robins, serving as vertebrate hosts. WNV and SLEV circulate in the same environments and have recently caused concurrent disease outbreaks in humans. The extent that coinfection of mosquitoes or birds may alter transmission dynamics, however, is not well characterized. We therefore sought to determine if coinfection alters infection kinetics and virus levels in birds and infection rates in mosquitoes. Accordingly, American robins (Turdus migratorius), two species of mosquitoes, and vertebrate and invertebrate cells were infected with WNV and/or SLEV to assess how simultaneous exposure may alter infection outcomes. There was variable impact of coinfection in vertebrate cells, with some evidence that SLEV can suppress WNV replication. However, robins had comparable viremia and antibody responses regardless of coinfection. Conversely, in Culex cells and mosquitoes, we saw a minimal impact of simultaneous exposure to both viruses on replication, with comparable infection, dissemination, and transmission rates in singly infected and coinfected mosquitoes. Importantly, while WNV and SLEV levels in coinfected mosquito midguts were positively correlated, we saw no correlation between them in salivary glands and saliva. These results reveal that while coinfection can occur in both avian and mosquito hosts, the viruses minimally impact one another. The potential for coinfection to alter virus population structure or the likelihood of rare genotypes emerging remains unknown.IMPORTANCEWest Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related viruses that are transmitted by the same mosquitoes and infect the same birds in nature. Both viruses circulate in the same regions and have caused concurrent outbreaks in humans. It is possible that mosquitoes, birds, and/or humans could be infected with both WNV and SLEV simultaneously, as has been observed with Zika, chikungunya, and dengue viruses. To study the impact of coinfection, we experimentally infected vertebrate and invertebrate cells, American robins, and two Culex species with WNV and/or SLEV. Robins were efficiently coinfected, with no impact of coinfection on virus levels or immune response. Similarly, in mosquitoes, coinfection did not impact infection rates, and mosquitoes could transmit both WNV and SLEV together. These results reveal that WNV and SLEV coinfection in birds and mosquitoes can occur in nature, which may impact public health and human disease risk.

Diverse single-stranded DNA viruses identified in New Zealand (Aotearoa) South Island robin (Petroica australis) fecal samples.

The South Island robin (Petroica australis) is a small passerine bird endemic to New Zealand (Aotearoa). Although its population has declined recently and it is considered 'at risk,' little research has been done to identify viruses in this species. This study aimed to survey the diversity of single-stranded DNA viruses associated with South Island robins in a small, isolated population on Nukuwaiata Island. In total, 108 DNA viruses were identified from pooled fecal samples collected from 38 individual robins sampled. These viruses belong to the Circoviridae (n = 10), Genomoviridae (n = 12), and Microviridae (n = 73) families. A number of genomes that belong to the phylum Cressdnaviricota but are otherwise unclassified (n = 13) were also identified. These results greatly expand the known viral diversity associated with South Island robins, and we identify a novel group of viruses most closely related genomoviruses.

Genomic monitoring to understand the emergence and spread of Usutu virus in the Netherlands, 2016-2018.

Usutu virus (USUV) is a mosquito-borne flavivirus circulating in Western Europe that causes die-offs of mainly common blackbirds (Turdus merula). In the Netherlands, USUV was first detected in 2016, when it was identified as the likely cause of an outbreak in birds. In this study, dead blackbirds were collected, screened for the presence of USUV and submitted to Nanopore-based sequencing. Genomic sequences of 112 USUV were obtained and phylogenetic analysis showed that most viruses identified belonged to the USUV Africa 3 lineage, and molecular clock analysis evaluated their most recent common ancestor to 10 to 4 years before first detection of USUV in the Netherlands. USUV Europe 3 lineage, commonly found in Germany, was less frequently detected. This analyses further suggest some extent of circulation of USUV between the Netherlands, Germany and Belgium, as well as likely overwintering of USUV in the Netherlands.

Usutu Virus Epizootic in Belgium in 2017 and 2018: Evidence of Virus Endemization and Ongoing Introduction Events.

Wildlife surveillance allowed the monitoring of the zoonotic mosquito-borne Usutu virus (USUV) in birds and bats (Pipistrellus pipistrellus) in southern Belgium in 2017 and 2018. USUV-RNA was detected in 69 birds (of 253) from 15 species, among which 7 species had not previously been reported to be susceptible to the infection. Similarly, 2 bats (of 10) were detected positive by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). USUV-associated lesions were mainly found in Eurasian Blackbirds (Turdus merula), in which USUV antigens were demonstrated by immunohistochemistry in the brain, heart, liver, kidney, intestine, and lung. Partial nonstructural protein 5 gene-based phylogenetic analysis showed several identical or closely related strains from 2016, 2017, and 2018 clustering together within Europe 3 or Africa 3 lineages. Further, one USUV strain detected in a common chaffinch (Fringilla coelebs) manifested a close genetic relationship with the European 1 strains circulating in Hungary and Austria. Our data provide evidence of USUV endemization in southern Belgium in local birds and bats, extension of the host range of the virus and ongoing virus introduction from abroad, likely by migratory birds. Our results highlight the need for vigilance in the forthcoming years toward new virus-associated outbreaks in birds and possible human infections in Belgium.

Focal amplification and suppression of West Nile virus transmission associated with communal bird roosts in northern Colorado.

To explain the patchy distribution of West Nile virus (WNV), we propose that avian immunity encountered by Culex vectors regulates WNV transmission, particularly at communal bird roosts. To test this hypothesis, we selected two test sites with communally roosting American robins (Turdus migratorius) and two control sites that lacked communal roosts. The density of vector-vertebrate contacts, represented by engorged Culex pipiens, was 23-fold greater at test sites compared to control sites, and the density of blood-engorged Cx. pipiens measured in resting mosquito traps correlated positively with the presence of robins and negatively with the presence of other birds, confirming an attraction to robins for blood feeding. WNV transmission was alternately up-regulated (amplification) and down-regulated (suppression) at both test sites. At one test site, infection in resting Cx. pipiens surged from zero to 37.2 per thousand within four weeks, and robin immunity rose from 8.4% to 64% before reducing to 33%. At this site, ten potentially infectious contacts between vector and vertebrates (including nine robins and a mourning dove [Zenaida macroura]) were documented. Infectious vector-vertebrate contacts were absent from control sites. The use of infectious vector-vertebrate contacts, rather than infected mosquitoes, to evaluate a transmission focus is novel.

Viral shedding of clade 2.3.4.4 H5 highly pathogenic avian influenza A viruses by American robins.

American robins (Turdus migratorius) are commonly associated with farmsteads in the United States and have shown previous evidence of exposure to an H5 avian influenza A virus (IAV) near a poultry production facility affected by a highly pathogenic (HP) H5 virus in Iowa, USA during 2015. We experimentally infected American robins with three clade 2.3.4.4 HP H5 viruses (H5N2 and H5N8). A total of 22/24 American robins shed virus, and all three strains were represented. The highest virus titres shed were 104.3 , 104.3 and 104.8 PFU/ml, associated respectively with viruses isolated from poultry, a captive gyrfalcon (Falco rusticolus), and a Northern pintail (Anas acuta). Of those birds that shed, viral shedding was initiated 1 or 2 days post-infection (DPI) and shedding ceased in all birds by 7 DPI. This study adds an additional synanthropic wildlife species to a growing list of animals that can successfully replicate and shed IAVs.

Breeding consequences of flavivirus infection in the collared flycatcher.

BACKGROUND: The breeding consequences of virus infections have rarely been studied in avian natural breeding populations. In this paper we investigated the links between humoral immunity following a natural flavivirus infection and reproduction in a wild bird population of collared flycatcher (Ficedula albicollis). We analyzed plasma from 744 birds for antibodies and correlated these results to a number of reproductive components. RESULTS: Nearly one third (27.8%) of the sampled collared flycatchers were found seropositive for flavivirus. Males had significantly more frequently flavivirus antibodies (32.3%) than females (25.1%). Seropositive females differed significantly from seronegative females in four traits: they had earlier lay date, higher body weight, higher survival rate and were older than seronegative females. The females did not differ in clutch size, number of fledged young or number of recruited young. Seropositive males had female partners with earlier lay date, i.e. the males bred earlier and they also produced more fledged young than seronegative males. In contrast, the males did not differ in clutch size, number of recruited young, male weight, age or survival. Interestingly, seropositive males had larger ornament, forehead badge size, than seronegative males. CONCLUSIONS: Collared flycatchers with an antibody response against flavivirus were more successful than birds with no antibody response, for any of the measured life history traits. The positive link between flavivirus antibody presence and life-history trait levels suggest that it is condition dependent in the collared flycatcher.

Stress hormones predict a host superspreader phenotype in the West Nile virus system.

Glucocorticoid stress hormones, such as corticosterone (CORT), have profound effects on the behaviour and physiology of organisms, and thus have the potential to alter host competence and the contributions of individuals to population- and community-level pathogen dynamics. For example, CORT could alter the rate of contacts among hosts, pathogens and vectors through its widespread effects on host metabolism and activity levels. CORT could also affect the intensity and duration of pathogen shedding and risk of host mortality during infection. We experimentally manipulated songbird CORT, asking how CORT affected behavioural and physiological responses to a standardized West Nile virus (WNV) challenge. Although all birds became infected after exposure to the virus, only birds with elevated CORT had viral loads at or above the infectious threshold. Moreover, though the rate of mortality was faster in birds with elevated CORT compared with controls, most hosts with elevated CORT survived past the day of peak infectiousness. CORT concentrations just prior to inoculation with WNV and anti-inflammatory cytokine concentrations following viral exposure were predictive of individual duration of infectiousness and the ability to maintain physical performance during infection (i.e. tolerance), revealing putative biomarkers of competence. Collectively, our results suggest that glucocorticoid stress hormones could directly and indirectly mediate the spread of pathogens.

Temporal Variation in Sindbis Virus Antibody Prevalence in Bird Hosts in an Endemic Area in Sweden.

Sindbis virus (SINV) is a mosquito-borne bird virus that occasionally causes human disease in Fennoscandia, suggested to have cyclic 7-year intervals between outbreaks. Reliable data on human infections in Sweden is however lacking. Here we investigated the SINV antibody prevalence among birds in a Swedish area endemic to SINV to scrutinize if a cyclic variation in antibody prevalence is present in the natural host of SINV. Serum from birds were sampled in the summers of 2002-2004 and 2009 in the floodplains of the River Dalälven in central Sweden, with 2002 and 2009 representing hypothesized years of SINV outbreaks. A total of 963 birds from 52 species (mainly passerines) were tested for the presence of SINV antibodies using a plaque reduction neutralization test. The highest SINV antibody prevalence was found in Turdidae species, specifically Fieldfare, Redwing and Song thrush in which more than 70% of sampled individuals had antibodies to SINV in 2009. The SINV antibody prevalence significantly varied between years with 2% in 2002, 8% in 2003, 14% in 2004 and 37% in 2009. Antibodies were found equally often in hatchlings and in adults and increased from early to late in the season. Clearly, the SINV antibody prevalence was not elevated in the bird hosts in the predicted outbreak year 2002, thus solid evidence of a cyclic occurrence of SINV in Sweden is still lacking.

A mathematical model for the spread of west nile virus in migratory and resident birds.

We develop a mathematical model for transmission of West Nile virus (WNV) that incorporates resident and migratory host avian populations and a mosquito vector population. We provide a detailed analysis of the model's basic reproductive number and demonstrate how the exposed infected, but not infectious, state for the bird population can be approximated by a reduced model. We use the model to investigate the interplay of WNV in both resident and migratory bird hosts. The resident host parameters correspond to the American Crow (Corvus brachyrhynchos), a competent host with a high death rate due to disease, and migratory host parameters to the American Robin (Turdus migratorius), a competent host with low WNV death rates. We find that yearly seasonal outbreaks depend primarily on the number of susceptible migrant birds entering the local population each season. We observe that the early growth rates of seasonal outbreaks is more influenced by the the migratory population than the resident bird population. This implies that although the death of highly competent resident birds, such as American Crows, are good indicators for the presence of the virus, these species have less impact on the basic reproductive number than the competent migratory birds with low death rates, such as the American Robins. The disease forecasts are most sensitive to the assumptions about the feeding preferences of North American mosquito vectors and the effect of the virus on the hosts. Increased research on the these factors would allow for better estimates of these important model parameters, which would improve the quality of future WNV forecasts.

Complete Genome Characterization of the Arumowot Virus (Unclassified Phlebovirus) Isolated from Turdus libonyanus Birds in the Central African Republic.

The Bunyaviridae family is currently composed of five genera, including Phlebovirus, in which several phleboviruses are associated with human diseases. Using high-throughput sequencing, we obtained and characterized one complete genome of the Arumowot virus (AMTV) isolated in 1978 from Turdus libonyanus, the Kurrichane Thrush, in the Central African Republic (CAR). The genomic segment of the new strain of AMTV isolated in the CAR had 75.4-83.5% sequence similarity and 82-98.4% amino acid similarity to the prototype sequence of AMTV. The different conserved proteins of the small (S) and large (L) segments (Nc, NSP, and RNA polymerase) showed close similarity at the amino acid level, whereas the polyprotein of the medium (M) segment was highly divergent, with 18% and 37.7%, respectively, for the prototype sequence of AMTV and the Odrenisrou virus (ODRV) isolated from Culex (Cx.) albiventris mosquitoes in the Tai forest, Ivory Coast. Phylogenetic analysis confirmed the sequence homology analysis and indicated that AMTV-CAR clustered into the Salehabad virus antigenic complex. The two closest viruses were the prototype sequences of AMTV originally isolated from Cx. antennatus mosquitoes and ODRV. These molecular data suggest the need for a deep genetic characterization of the diversity of this viral species to enhance its detection in the Central African region and to understand better its behavior and life cycle so that its potential spread to the human population can be prevented.

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.

The complete sequence of a West Nile virus lineage 2 strain detected in a Hyalomma marginatum marginatum tick collected from a song thrush (Turdus philomelos) in eastern Romania in 2013 revealed closest genetic relationship to strain Volgograd 2007.

In this study the first complete sequence of the West Nile virus (WNV) lineage 2 strain currently circulating in Romania was determined. The virus was detected in a Hyalomma marginatum marginatum tick collected from a juvenile song thrush (Turdus philomelos) in the Romanian Danube Delta close to the city of Tulcea, end of August 2013. Our finding emphasizes the role of ticks in introduction and maintenance of WNV infections. Sequence analyses revealed close genetic relationship of the Romanian WNV strain to strain Reb_Volgograd_07_H, which was isolated from human brain tissue during an outbreak of West Nile neuroinvasive disease (WNND) in Russia in 2007. In 2010 the Eastern European lineage 2 WNV caused an outbreak of human WNND in Romania. Partial sequences from subsequent years demonstrated that this WNV strain became endemic in Eastern Europe and has been causing outbreaks of varying sizes in southern Russia since 2007 and in Romania since 2010.

Host group formation decreases exposure to vector-borne disease: a field experiment in a 'hotspot' of West Nile virus transmission.

Animals can decrease their individual risk of predation by forming groups. The encounter-dilution hypothesis extends the potential benefits of gregariousness to biting insects and vector-borne disease by predicting that the per capita number of insect bites should decrease within larger host groups. Although vector-borne diseases are common and can exert strong selective pressures on hosts, there have been few tests of the encounter-dilution effect in natural systems. We conducted an experimental test of the encounter-dilution hypothesis using the American robin (Turdus migratorius), a common host species for the West Nile virus (WNV), a mosquito-borne pathogen. By using sentinel hosts (house sparrows, Passer domesticus) caged in naturally occurring communal roosts in the suburbs of Chicago, we assessed sentinel host risk of WNV exposure inside and outside of roosts. We also estimated per capita host exposure to infected vectors inside roosts and outside of roosts. Sentinel birds caged inside roosts seroconverted to WNV more slowly than those outside of roosts, suggesting that social groups decrease per capita exposure to infected mosquitoes. These results therefore support the encounter-dilution hypothesis in a vector-borne disease system. Our results suggest that disease-related selective pressures on sociality may depend on the mode of disease transmission.

Parasites in the city: degree of urbanization predicts poxvirus and coccidian infections in house finches (Haemorhous mexicanus).

BACKGROUND: Urbanization can strongly impact the physiology, behavior, and fitness of animals. Conditions in cities may also promote the transmission and success of animal parasites and pathogens. However, to date, no studies have examined variation in the prevalence or severity of several distinct pathogens/parasites along a gradient of urbanization in animals or if these infections increase physiological stress in urban populations. METHODOLOGY/PRINCIPAL FINDINGS: Here, we measured the prevalence and severity of infection with intestinal coccidians (Isospora sp.) and the canarypox virus (Avipoxvirus) along an urban-to-rural gradient in wild male house finches (Haemorhous mexicanus). In addition, we quantified an important stress indicator in animals (oxidative stress) and several axes of urbanization, including human population density and land-use patterns within a 1 km radius of each trapping site. Prevalence of poxvirus infection and severity of coccidial infection were significantly associated with the degree of urbanization, with an increase of infection in more urban areas. The degrees of infection by the two parasites were not correlated along the urban-rural gradient. Finally, levels of oxidative damage in plasma were not associated with infection or with urbanization metrics. CONCLUSION/SIGNIFICANCE: These results indicate that the physical presence of humans in cities and the associated altered urban landscape characteristics are associated with increased infections with both a virus and a gastrointestinal parasite in this common songbird resident of North American cities. Though we failed to find elevations in urban- or parasite/pathogen-mediated oxidative stress, humans may facilitate infections in these birds via bird feeders (i.e. horizontal disease transmission due to unsanitary surfaces and/or elevations in host population densities) and/or via elevations in other forms of physiological stress (e.g. corticosterone, nutritional).

Possible role of songbirds and parakeets in transmission of influenza A(H7N9) virus to humans.

Avian-origin influenza A(H7N9) recently emerged in China, causing severe human disease. Several subtype H7N9 isolates contain influenza genes previously identified in viruses from finch-like birds. Because wild and domestic songbirds interact with humans and poultry, we investigated the susceptibility and transmissibility of subtype H7N9 in these species. Finches, sparrows, and parakeets supported replication of a human subtype H7N9 isolate, shed high titers through the oropharyngeal route, and showed few disease signs. Virus was shed into water troughs, and several contact animals seroconverted, although they shed little virus. Our study demonstrates that a human isolate can replicate in and be shed by such songbirds and parakeets into their environment. This finding has implications for these birds' potential as intermediate hosts with the ability to facilitate transmission and dissemination of A(H7N9) virus.

Vector contact rates on Eastern bluebird nestlings do not indicate West Nile virus transmission in Henrico County, Virginia, USA.

Sensitive indicators of spatial and temporal variation in vector-host contact rates are critical to understanding the transmission and eventual prevention of arboviruses such as West Nile virus (WNV). Monitoring vector contact rates on particularly susceptible and perhaps more exposed avian nestlings may provide an advanced indication of local WNV amplification. To test this hypothesis we monitored WNV infection and vector contact rates among nestlings occupying nest boxes (primarily Eastern bluebirds; Sialia sialis, Turdidae) across Henrico County, Virginia, USA, from May to August 2012. Observed host-seeking rates were temporally variable and associated with absolute vector and host abundances. Despite substantial effort to monitor WNV among nestlings and mosquitoes, we did not detect the presence of WNV in these populations. Generally low vector-nestling host contact rates combined with the negative WNV infection data suggest that monitoring transmission parameters among nestling Eastern bluebirds in Henrico County, Virginia, USA may not be a sensitive indicator of WNV activity.

Partial genetic characterization of Sedlec virus (Orthobunyavirus, Bunyaviridae).

Sedlec virus (SEDV) was isolated from the blood of a reed warbler (Acrocephalus scirpaceus) in July 1984 in South Moravia, Czech Republic. In this study first genetic data of SEDV are presented which allow an estimate on its phylogenetic and taxonomic positioning within the genus Orthobunyavirus. The phylogenetic analysis of a 369 nt long stretch within the S segment (nucleocapsid protein gene and non-structural S protein gene) indicates genetic relatedness of SEDV to Leanyer virus and Simbu group viruses, while the phylogenetic tree based on 1796nt long sequences of the L segment (RNA-dependent RNA polymerase gene) demonstrates genetic relationship of SEDV to two yet unclassified orthobunyaviruses: I612045 virus (isolated in India in 1961) and Oyo virus (isolated in Nigeria in 1964). Considering the genetic distances and the phylogenetic analyses, SEDV might represent a novel serogroup of the Orthobunyavirus genus.

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.