Molecular characterization of an avian rotavirus a strain detected from a large-billed crow (Corvus macrorhynchos) in Japan.

Avian rotaviruses A (RVAs) are occasionally transmitted to animals other than the original hosts across species barriers. Information on RVAs carried by various bird species is important for identifying the origin of such interspecies transmission. In this study, to facilitate an understanding of the ecology of RVAs from wild birds, we characterized all of the genes of an RVA strain, JC-105, that was detected in a fecal sample of a large-billed crow (Corvus macrorhynchos) in Japan. All of the genes of this strain except for the VP4 and VP7 genes, which were classified as novel genotypes (P[56] and G40, respectively), were closely related to those of the avian-like RVA strain detected from a raccoon, indicating the possibility that crows had been involved in the transmission of avian RVAs to raccoons. Our findings highlight the need for further viral investigations in wild birds and mammals to understand the mechanisms of avian-to-mammal RVA transmission.

A multi-species, multi-pathogen avian viral disease outbreak event: Investigating potential for virus transmission at the wild bird - poultry interface.

A free-range organic broiler (Gallus gallus domesticus) premises in Staffordshire was infected by high pathogenicity avian influenza virus (HPAIV) H5N8 during the 2020-2021 epizootic in the United Kingdom (UK). Following initial confirmation of the infection in poultry, multiple wild bird species were seen scavenging on chicken carcasses. Detected dead wild birds were subsequently demonstrated to have been infected and succumbed to HPAIV H5N8. Initially, scavenging species, magpie (Pica pica) and raven (Corvus corax) were found dead on the premises but over the following days, buzzards (Buteo buteo) were also found dead within the local area with positive detection of HPAIV in submitted carcasses. The subacute nature of microscopic lesions within a buzzard was consistent with the timeframe of infection. Finally, a considerable number of free-living pheasants (Phasianus colchicus) were also found dead in the surrounding area, with carcasses having higher viral antigen loads compared to infected chickens. Limited virus dissemination was observed in the carcasses of the magpie, raven, and buzzard. Further, an avirulent avian paramyxovirus type 1 (APMV-1) was detected within poultry samples as well as in the viscera of a magpie infected with HPAIV. Immunohistochemistry did not reveal colocalization of avian paramyxovirus antigens with lesions, supporting an avirulent APMV-1 infection. Overall, this case highlights scenarios in which bi-directional transmission of avian viral diseases between commercial and wild bird species may occur. It also underlines the importance of bio separation and reduced access when infection pressure from HPAIV is high.

Mosquito blood-feeding patterns and nesting behavior of American crows, an amplifying host of West Nile virus.

BACKGROUND: Although American crows are a key indicator species for West Nile virus (WNV) and mount among the highest viremias reported for any host, the importance of crows in the WNV transmission cycle has been called into question because of their consistent underrepresentation in studies of Culex blood meal sources. Here, we test the hypothesis that this apparent underrepresentation could be due, in part, to underrepresentation of crow nesting habitat from mosquito sampling designs. Specifically, we examine how the likelihood of a crow blood meal changes with distance to and timing of active crow nests in a Davis, California, population. METHODS: Sixty artificial mosquito resting sites were deployed from May to September 2014 in varying proximity to known crow nesting sites, and Culex blood meal hosts were identified by DNA barcoding. Genotypes from crow blood meals and local crows (72 nestlings from 30 broods and 389 local breeders and helpers) were used to match mosquito blood meals to specific local crows. RESULTS: Among the 297 identified Culex blood meals, 20 (6.7%) were attributable to crows. The mean percentage of blood meals of crow origin was 19% in the nesting period (1 May-18 June 2014), but 0% in the weeks after fledging (19 June-1 September 2014), and the likelihood of a crow blood meal increased with proximity to an active nest: the odds that crows hosted a Culex blood meal were 38.07 times greater within 10 m of an active nest than > 10 m from an active nest. Nine of ten crow blood meals that could be matched to a genotype of a specific crow belonged to either nestlings in these nests or their mothers. Six of the seven genotypes that could not be attributed to sampled birds belonged to females, a sex bias likely due to mosquitoes targeting incubating or brooding females. CONCLUSION: Data herein indicate that breeding crows serve as hosts for Culex in the initial stages of the WNV spring enzootic cycle. Given their high viremia, infected crows could thereby contribute to the re-initiation and early amplification of the virus, increasing its availability as mosquitoes shift to other moderately competent later-breeding avian hosts.

WEST NILE VIRUS INFECTION IN RUFFED GROUSE (BONASA UMBELLUS) IN PENNSYLVANIA, USA: A MULTI-YEAR COMPARISON OF STATEWIDE SEROSURVEYS AND VECTOR INDICES.

Eastern populations of Ruffed Grouse (Bonasa umbellus) have been in a decades-long decline across the mid-Atlantic and southern Appalachian Mountains of the US. West Nile virus (WNV), which first arrived in the US in 1999, is suspected to have contributed to these declines based on decreased population indices since the arrival of WNV in Pennsylvania as well as on high, experimentally induced WNV-associated morbidity rates. A 3-yr statewide survey was conducted across Pennsylvania to measure flavivirus (i.e., WNV) seroprevalence among hunter-harvested grouse. The overall seroprevalence from 2015-17 was 14.4% (81/563); annual seroprevalence ranged from 2.8% (4/145) in the 2017 hunt year to 22.6% (52/230) in 2016-17. We analyzed the effects of numerous variables (i.e., Ruffed Grouse age and sex, hunt year, WNV vector index [VI], and region of Pennsylvania) on WNV serostatus by logistic regression. While there was no significant difference in WNV seroprevalence between sex and age group, there was significant variation in seroprevalence between geographic regions of Pennsylvania and across hunt years. Additionally, there was a negative correlation between WNV seroprevalence and VI. Low seroprevalence rates among Ruffed Grouse corresponded to years with a high VI, supporting experimental findings that Ruffed Grouse may be highly susceptible to WNV-associated disease. Additional strategic research efforts are essential to more effectively measure the effects of WNV on Ruffed Grouse and other vulnerable avian species.

Molecular characterization of the re-emerging West Nile virus in avian species and equids in Israel, 2018, and pathological description of the disease.

BACKGROUND: In this report we describe the molecular and pathological characteristics of West Nile virus (WNV) infection that occurred during the summer and fall of 2018 in avian species and equines. WNV is reported in Israel since the 1950s, with occasional outbreaks leading to significant morbidity and mortality in birds, high infection in horses and humans, and sporadic fatalities in humans. METHODS: Animal and avian carcasses in a suitable condition were examined by post-mortem analysis. Tissue samples were examined for WNV by RT-qPCR and the viral load was quantified. Samples with sufficient material quality were further analyzed by Endpoint PCR and sequencing, which was used for phylogenetic analysis. Tissue samples from positive animals were used for culturing the virus in Vero and C6/36 cells. RESULTS: WNV RNA was detected in one yellow-legged gull (Larus michahellis), two long-eared owls (Asio otus), two domesticated geese (Anser anser), one pheasant (Phasianus colchicus), four hooded crows (Corvus cornix), three horses and one donkey. Pathological and histopathological findings were characteristic of viral infection. Molecular analysis and viral load quantification showed varying degrees of infection, ranging between 70-1.4 × 106 target copies per sample. Phylogenetic analysis of a 906-bp genomic segment showed that all samples belonged to Lineage 1 clade 1a, with the following partition: five samples from 2018 and one sample detected in 2016 were of Cluster 2 Eastern European, two of Cluster 2 Mediterranean and four of Cluster 4. Four of the positive samples was successfully propagated in C6/36 and Vero cell lines for further work. CONCLUSIONS: WNV is constantly circulating in wild and domesticated birds and animals in Israel, necessitating constant surveillance in birds and equines. At least three WNV strains were circulating in the suspected birds and animals examined. Quantitative analysis showed that the viral load varies significantly between different organs and tissues of the infected animals.

Evaluation of potential risk of transmission of avian influenza A viruses at live bird markets in response to unusual crow die-offs in Bangladesh.

In response to unusual crow die-offs from avian influenza A(H5N1) virus infection during January-February 2017 in Dhaka, Bangladesh, a One Health team assessed potential infection risks in live bird markets (LBMs). Evidence of aerosolized avian influenza A viruses was detected in LBMs and in the respiratory tracts of market workers, indicating exposure and potential for infection. This study highlighted the importance of surveillance platforms with a coordinated One Health strategy to investigate and mitigate zoonotic risk.

Detection of West Nile virus by real-time PCR in crows in northern provinces of Iran.

BACKGROUND & OBJECTIVES: West Nile virus (WNV) is a positive-sense, single-stranded RNA virion, that belongs to the Flaviviridae family. This virus is preserved in a bird-mosquito cycle that is capable of inducing diseases as a dead-end or endpoint host in humans as well as horses. In 2016, a suspicious case of crow population death was reported by the Department of Environment, Ministry of Health, Iran. Considering the mass migration of birds together with the WNV-related symptoms, including uncoordinated walking, ataxia, inability to fly, lack of awareness, and abnormal body posture, it was necessary to further investigate the possible causes of this incident. The objective of this study was molecular detection of WNV in crows utilizing the real-time PCR method in the northern provinces of Iran. METHODS: A total of 12 crows (8 dead, 4 alive) with a possible WNV infection, were collected from the northern provinces of Iran (Golestan, Mazandaran, and Guilan). A tissue sample of the liver, kidney, or lung was collected from all the crows, and RNA was isolated using an RNA extraction kit. A one-step real-time PCR method using a TaqMan probe was used for virus detection. RESULTS: All the infected crows were positive for WNV. The 132-bp real-time PCR amplicon of the genome was detected in all the samples. Comparative phylogenetic analysis revealed that WNV isolated from Iran clustered with strains from the USA, Hungary, and Culex pipiens. INTERPRETATION & CONCLUSION: The WNV genome sequence was detected in all the infected crows. The results confirmed the connection of this isolation with clade1a strains. Hence, determining the epidemiologic and prevalence characteristics of the WNV for transmission control is of critical importance in Iran.

Complete genomic sequence of crow-dipper mosaic-associated virus, a novel macluravirus infecting Pinellia ternata.

A new macluravirus infecting Pinellia ternata in China was identified by high-throughput sequencing (HTS) and tentatively named "crow-dipper mosaic-associated virus" (CrdMV). The complete genome sequence of CrdMV was determined by reverse transcription (RT) PCR and rapid amplification of cDNA ends (RACE) PCR. The genomic RNA of CrdMV consists of 8,454 nucleotides (nt), excluding the poly(A) tail at the 3' end. CrdMV has a genomic structure typical of macluraviruses, with large open reading frame encoding a polyprotein of 2,696 amino acids (aa). CrdMV shares 54.40%-59.37% nt sequence identity at the genome sequence level, 48.00%-58.58% aa sequence identity, at the polyprotein sequence level and 37.27%-49.22% aa sequence identity at the CP sequence level with other members of the genus Macluravirus. These values are well below the species demarcation threshold for the family Potyviridae. Phylogenetic analysis based on the amino acid sequences of polyproteins confirmed that CrdMV clusters closely with broad-leafed dock virus A (BDVA, GenBank accession no. KU053507). These results suggest that CrdMV should be considered a distinct member of the genus Macluravirus.

The potential role of scavengers in spreading African swine fever among wild boar.

Understanding the transmission patterns of African swine fever (ASF) among wild boar (Sus scrofa) is an issue of major interest, especially in the wake of the current ASF epidemic. Given the high stability of ASF-virus, there is concern about scavengers spreading infectious carcass material in the environment. Here, we describe scavenging activities on 32 wild boar carcasses in their natural habitat in Germany. Using digital cameras, we detected 22 vertebrates at the study sites, thereof two mammal and three bird species scavenging. The most frequently detected species was the raccoon dog Nyctereutes procyonoides (44% of all visits). Raccoon dogs, red foxes (Vulpes vulpes), and buzzards (Buteo buteo) scavenged in the warm and the cold season, while ravens (Corvus corax) and white-tailed eagles (Haliaeetus albicilla) scavenged only in the cold season. In summer, however, insects removed most of the carcass biomass. Although most of the material was consumed on the spot, foxes, raccoon dogs and ravens left the study sites in rare cases with a small piece of meat in their mouths or beaks. We conclude that scavengers represent a minor risk factor for spreading ASF, but may contribute to reducing local virus persistence by metabolizing infected carcasses.

Full genome characterization of Iranian H5N8 highly pathogenic avian influenza virus from Hooded Crow (Corvus cornix), 2017: The first report.

During 2014-2017 Clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses (HPAIVs) have spread worldwide. In 2016, an epidemic of HPAIV H5N8 in Iran caused mass deaths among wild birds, and several commercial poultry farms and captive bird holdings were affected and continue to experience problems. Several outbreaks were reported in 2017. One of them is related to Hooded crow (Corvus cornix) in a national park in Esfahan province in 2017. Whole genome sequencing and characterization have been done on the detected H5N8 sample. Based on HA sequencing results, it belongs to 2.3.4.4 clade, and the cleavage site is (PLREKRRKR/G). Phylogenetic analysis of the HA gene showed that the Iran 2017 H5N8 virus clustered within subgroup Russia 2016 2.3.4.4 b of group B in H5 clade 2.3.4.4 HPAIV. On the other hand, the NA gene of the virus is placed in group C of Eurasian lineage. Complete genome characterization of this virus revealed probable reassortment of the virus with East-Asian low-pathogenic influenza viruses. Furthermore, the virus possessed some phenotypic markers related to the increased potential for transmission and pathogenicity to mammals at internal segments. This study is the first full genome characterization H5N8 HPAIV in Iran. The data complete the puzzle of molecular epidemiology of H5N8 HPAIV in Iran and the region. Our study provides evidence for fast and continuing reassortment of H5 clade 2.3.4.4 viruses, that might lead to changes in virus structural and functional characteristics such as the route and method of transmission of the virus and virus infective, pathogenic and zoonotic potential.

Overwintering of West Nile virus in a bird community with a communal crow roost.

In temperate climates, transmission of West Nile virus (WNV) is detectable rarely during the coldest months (late fall through early spring), yet the virus has reappeared consistently during the next warm season. Several mechanisms may contribute to WNV persistence through winter, including bird-to-bird transmission among highly viremic species. Here we consider whether, under realistic scenarios supported by field and laboratory evidence, a winter bird community could sustain WNV through the winter in the absence of mosquitoes. With this purpose we constructed a deterministic model for a community of susceptible birds consisting of communally roosting crows, raptors and other birds. We simulated WNV introduction and subsequent transmission dynamics during the winter under realistic initial conditions and model parameterizations, including plausible contact rates for roosting crows. Model results were used to determine whether the bird community could yield realistic outbreaks that would result in WNV infectious individuals at the end of the winter, which would set up the potential for onward horizontal transmission into summer. Our findings strongly suggest that winter crow roosts could allow for WNV persistence through the winter, and our model results provide synthesis to explain inconclusive results from field studies on WNV overwintering in crow roosts.

West Nile and St. Louis encephalitis viral genetic determinants of avian host competence.

West Nile virus (WNV) and St. Louis encephalitis (SLEV) virus are enzootically maintained in North America in cycles involving the same mosquito vectors and similar avian hosts. However, these viruses exhibit dissimilar viremia and virulence phenotypes in birds: WNV is associated with high magnitude viremias that can result in mortality in certain species such as American crows (AMCRs, Corvus brachyrhynchos) whereas SLEV infection yields lower viremias that have not been associated with avian mortality. Cross-neutralization of these viruses in avian sera has been proposed to explain the reduced circulation of SLEV since the introduction of WNV in North America; however, in 2015, both viruses were the etiologic agents of concurrent human encephalitis outbreaks in Arizona, indicating the need to re-evaluate host factors and cross-neutralization responses as factors potentially affecting viral co-circulation. Reciprocal chimeric WNV and SLEV viruses were constructed by interchanging the pre-membrane (prM)-envelope (E) genes, and viruses subsequently generated were utilized herein for the inoculation of three different avian species: house sparrows (HOSPs; Passer domesticus), house finches (Haemorhous mexicanus) and AMCRs. Cross-protective immunity between parental and chimeric viruses were also assessed in HOSPs. Results indicated that the prM-E genes did not modulate avian replication or virulence differences between WNV and SLEV in any of the three avian species. However, WNV-prME proteins did dictate cross-protective immunity between these antigenically heterologous viruses. Our data provides further evidence of the important role that the WNV / SLEV viral non-structural genetic elements play in viral replication, avian host competence and virulence.

A geographic study of West Nile virus in humans, dead corvids and mosquitoes in Ontario using spatial scan statistics with a survival time application.

Surveillance of West Nile virus (WNv) in Ontario has included passive reporting of human cases and testing of trapped mosquitoes and dead birds found by the public. The dead bird surveillance programme was limited to testing within a public health unit (PHU) until a small number of birds test positive. These dead corvid and mosquito surveillance programmes have not been compared for their ability to provide early warning in geographic areas where human cases occur each year. Spatial scan statistics were applied to time-to-event survival data based on first cases of WNv in found dead corvids, mosquitoes and humans. Clusters identified using raw data were compared to clusters based on model-adjusted survival times to evaluate whether geographic and sociodemographic factors influenced their distribution. Statistically significant (p < .05) space-time clusters of PHUs with faster time to detection were found using each surveillance data stream. During 2002-2004, the corvid surveillance programme outperformed the mosquito programme in terms of time to WNv detection, while the clusters of first-positive mosquito pools were more spatially similar to first human cases. In 2006, a cluster of first-positive dead corvids was located in northern PHUs and preceded a cluster of early human cases that was identified after controlling for the influence of geographic region and sociodemographic profile.

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.

AN EPIZOOTIC OF EMERGING NOVEL AVIAN POX IN CARRION CROWS (CORVUS CORONE) AND LARGE-BILLED CROWS (CORVUS MACRORHYNCHOS) IN JAPAN.

In 2006-10, an epizootic of emerging avian pox occurred in Carrion Crows ( Corvus corone ) and Large-billed Crows ( Corvus macrorhynchos ), leading to mortality of juvenile crows in Hokkaido, the northernmost island of Japan. We diagnosed 27 crows with proliferative skin lesions (19 carcasses and eight biopsied cases [one in zoo captivity]) as avian pox clinically, histopathologically by detection of Avipoxvirus-specific 4b core protein (P4b) gene, and epidemiologically. The fatal cases demonstrated intensively severe infection and aggressive lesions with secondary bacterial infection. Since the first identification of avian pox in Sapporo, Japan, in 2006, the frequency of mortality events has increased, peaking in 2007-08. Mortalities have subsequently occurred in other areas, suggesting disease expansion. In Sapporo, prevalence of avian pox evaluated by field censuses during 2007-12 was 17.6% (6.6-27.2%), peaked during 2007-08 and 2008-09, and then decreased. All diseased crows were juveniles, except for one adult. The number of crows assembling in the winter roosts had been stable for >10 yr; however, it declined in 2007-08, decreased by about 50% in 2008-09, and recovered to the previous level in 2009-10, correlated with the avian pox outbreak. Thus, avian pox probably contributed to the unusual crow population decline. All P4b sequences detected in six specimens in Sapporo were identical and different from any previously reported sequences. The sequence detected in the zoo-kept crow was distinct from any reported clades, and interspecies transmission was suspected. This report demonstrates an emerging novel avian pox in the Japanese avifauna and in global populations of Carrion Crows and Large-billed Crows. Longitudinal monitoring is needed to evaluate its impact on the crow population.

A comparison of West Nile virus surveillance using survival analyses of dead corvid and mosquito pool data in Ontario, 2002-2008.

The aim of this study was to improve understanding of the relative performance of the use of dead wild corvids and mosquito pools infected with West Nile virus (WNv) in surveillance for WNv activity in the environment. To this end, all records on dead corvid submissions and mosquito pools tested in Public Health Units (PHUs) in Ontario, from 2002 to 2008, were explored. Survival analyses were employed using the first-WNv-positive cases detected each year for each PHU, and censored observations for PHUs which did not detect WNv during a given year using each data source (504 observations). Survival analyses were employed to compare the number of surveillance weeks before WNv was detected by either data source, and the influence of temporal, geographic and sociodemographic factors on these data. The outcome measurement for the final accelerated failure time (AFT) model with log-logistic distribution was a time ratio, which represents the ratio of the survival time of one group relative to another. Dead corvid surveillance was faster at detecting WNv than testing mosquito pools during the early years of WNv incursion into Ontario, while mosquito testing found WNv more quickly later in the study period. There was also regional variation in time-to-detection of WNv, by modality, as well as for various types of urban/rural settings. In comparison to mosquito surveillance, West Nile virus was detected more quickly using dead corvid surveillance in sparsely populated regions. These areas may benefit from collection of dead corvids to optimize detection and direct early surveillance efforts. When we compared the time-to-detection of WNv using dead corvids and the onset of human cases in PHUs, we found that dead corvid surveillance was predictive of West Nile activity in health units that reported human cases during the first 3 years of the incursion into Ontario.

West Nile Virus Activity in a Winter Roost of American Crows (Corvus brachyrhynchos): Is Bird-To-Bird Transmission Important in Persistence and Amplification?

Since its emergence in North America, West Nile virus (WNV) has had a large impact on equines, humans, and wild bird communities, yet gaps remain in our understanding of how the virus persists at temperate latitudes when winter temperatures preclude virus replication and host-seeking activity by mosquito vectors. Bird-to-bird transmission at large communal American Crow roosts could provide one mechanism for WNV persistence. Herein, we describe seasonal patterns of crow and Culex mosquito abundance, WNV infection rates, and the prevalence of WNV-positive fecal samples at a winter crow roost to test the hypothesis that bird-to-bird transmission allows WNV to persist at winter crow roosts. Samples were collected from large winter crow roosts in the Sacramento Valley of California from January 2013 until August 2014, encompassing two overwintering roost periods. West Nile virus RNA was detected in local crow carcasses in both summer [13/18 (72% WNV positive)] and winter [18/44 (41% WNV positive)] 2013-2014. Winter infections were unlikely to have arisen by recent bites from infected mosquitoes because Culex host-seeking activity was very low in winter and all Culex mosquitoes collected during winter months tested negative for WNV. Opportunities existed for fecal-oral transfer at the overwintering roost: most carcasses that tested positive for WNV had detectable viral RNA in both kidney and cloacal swabs, suggesting that infected crows were shedding virus in their feces, and >50% of crows at the roost were stained with feces by mid-winter. Moreover, 2.3% of fecal samples collected in late summer, when mosquitoes were active, tested positive for WNV RNA. Nevertheless, none of the 1,119 feces collected from three roosts over two winters contained detectable WNV RNA. This study provided evidence of WNV infection in overwintering American crows without mosquito vector activity, but did not elucidate a mechanism of WNV transmission during winter.

Next-generation sequencing shows West Nile virus quasispecies diversification after a single passage in a carrion crow (Corvus corone) in vivo infection model.

West Nile virus (WNV) occurs as a population of genetic variants (quasispecies) infecting a single animal. Previous low-resolution viral genetic diversity estimates in sampled wild birds and mosquitoes, and in multiple-passage adaptation studies in vivo or in cell culture, suggest that WNV genetic diversification is mostly limited to the mosquito vector. This study investigated genetic diversification of WNV in avian hosts during a single passage using next-generation sequencing. Wild-captured carrion crows were subcutaneously infected using a clonal Middle-East WNV. Blood samples were collected 2 and 4 days post-infection. A reverse-transcription (RT)-PCR approach was used to amplify the WNV genome directly from serum samples prior to next-generation sequencing resulting in an average depth of at least 700 × in each sample. Appropriate controls were sequenced to discriminate biologically relevant low-frequency variants from experimentally introduced errors. The WNV populations in the wild crows showed significant diversification away from the inoculum virus quasispecies structure. By contrast, WNV populations in intracerebrally infected day-old chickens did not diversify from that of the inoculum. Where previous studies concluded that WNV genetic diversification is only experimentally demonstrated in its permissive insect vector species, we have experimentally shown significant diversification of WNV populations in a wild bird reservoir species.

Susceptibility of Carrion Crows to Experimental Infection with Lineage 1 and 2 West Nile Viruses.

West Nile virus (WNV) outbreaks in North America have been characterized by substantial die-offs of American crows (Corvus brachyrhynchos). In contrast, a low incidence of bird deaths has been observed during WNV epidemic activity in Europe. To examine the susceptibility of the western European counterpart of American crows, we inoculated carrion crows (Corvus corone) with WNV strains isolated in Greece (Gr-10), Italy (FIN and Ita09), and Hungary (578/10) and with the highly virulent North American genotype strain (NY99). We also inoculated American crows with a selection of these strains to examine the strains' virulence in a highly susceptible bird species. Infection with all strains, except WNV FIN, resulted in high rates of death and high-level viremia in both bird species and virus dissemination to several organs. These results suggest that carrion crows are highly susceptible to WNV and may potentially be useful as part of dead bird surveillance for early warning of WNV activity in Europe.

An ex vivo avian leukocyte culture model for West Nile virus infection.

West Nile virus (WNV) replicates in a wide variety of avian species, which act as amplification hosts. In particular, WNV generates high titers and elicits severe pathology in American crows (AMCRs; Corvus brachyrhynchos), a species that has been used as a sentinel for WNV transmission. Although the specific cellular targets of WNV replication in AMCRs are not well defined, preliminary evidence suggests that leukocytes may be an important target of early replication. Therefore, development of a protocol for ex vivo culture of AMCR leukocytes as a model for assessing differential avian host susceptibility is described herein. WNV growth in these cultures mirrored in vivo viremia profiles. These data indicate that ex vivo leukocyte cultures can be used for preliminary pathological assessment of novel WNV strains and potentially of other flaviviruses that use avian reservoir hosts.