Richard D. Slemons

The Evolutionary Genetics and Emergence of Avian Influenza Viruses in Wild Birds

We surveyed the genetic diversity among avian influenza virus (AIV) in wild birds, comprising 167 complete viral genomes from 14 bird species sampled in four locations across the United States. These isolates represented 29 type A influenza virus hemagglutinin (HA) and neuraminidase (NA) subtype combinations, with up to 26% of isolates showing evidence of mixed subtype infection. Through a phylogenetic analysis of the largest data set of AIV genomes compiled to date, we were able to document a remarkably high rate of genome reassortment, with no clear pattern of gene segment association and occasional inter-hemisphere gene segment migration and reassortment. From this, we propose that AIV in wild birds forms transient “genome constellations,” continually reshuffled by reassortment, in contrast to the spread of a limited number of stable genome constellations that characterizes the evolution of mammalian-adapted influenza A viruses.

Type-A influenza viruses isolated from wild free-flying ducks in California.

From 6 October 1972 to 3 December 1972, 41 type-A influenza virus isolants were recovered from free-flying wild ducks, and 7 isolants from domestic ducks in southern California. The typespecific antigen (ribonucleoprotein) was identified by the agar-geldiffusion test, and tentative identification of one strain-specific antigen (hemagglutinin) was attempted by the hemagglutinationinhibition test. These isolants support the theories that wild birds play an important role in the dissemination of type-A influenza viruses and may provide optimum conditions for genetic interaction of type-A influenza viruses, resulting in new hybrid strains.

Phylogenetic Diversity among Low-Virulence Newcastle Disease Viruses from Waterfowl and Shorebirds and Comparison of Genotype Distributions to Those of Poultry-Origin Isolates

ABSTRACT Low-virulence Newcastle disease viruses (loNDV) are frequently recovered from wild bird species, but little is known about their distribution, genetic diversity, or potential to cause disease in poultry. NDV isolates recovered from cloacal samples of apparently healthy waterfowl and shorebirds (WS) in the United States during 1986 to 2005 were examined for genomic diversity and their potential for virulence (n = 249). In addition 19 loNDV isolates from U.S. live bird markets (LBMs) were analyzed and found to be genetically distinct from NDV used in live vaccines but related to WS-origin NDV. Phylogenetic analysis of the fusion protein identified nine novel genotypes among the class I NDV, and new genomic subgroups were identified among genotypes I and II of the class II viruses. The WS-origin viruses exhibited broad genetic and antigenic diversity, and some WS genotypes displayed a closer phylogenetic relationship to LBM-origin NDV. All NDV were predicted to be lentogenic based upon sequencing of the fusion cleavage site, intracerebral pathogenicity index, or mean death time in embryo assays. The USDA real-time reverse transcription-PCR assay, which targets the matrix gene, identified nearly all of the class II NDV tested but failed to detect class I viruses from both LBM and WS. The close phylogenetic proximity of some WS and LBM loNDV suggests that viral transmission may occur among wild birds and poultry; however, these events may occur unnoticed due to the broad genetic diversity of loNDV, the lentogenic presentation in birds, and the limitations of current rapid diagnostic tools.

Using RRT-PCR analysis and virus isolation to determine the prevalence of avian influenza virus infections in ducks at Minto Flats State Game Refuge, Alaska, during August 2005.

SummaryThis study describes surveillance for avian influenza viruses (AIV) in the Minto Flats State Game Refuge, high-density waterfowl breeding grounds in Alaska. Five hundred paired cloacal samples from dabbling ducks (Northern Pintail, Mallard, Green Wing Teal, and Widgeon) were placed into ethanol and viral transport medium (VTM). Additional ethanol-preserved samples were taken. Of the ethanol-preserved samples, 25.6% were AIV RNA-positive by real-time RT-PCR. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined for 38 of the first-passage isolates, and four first-passage isolates could not be definitively subtyped. Five influenza A virus HA–NA combinations were identified: H3N6, H3N8, H4N6, H8N4, and H12N5. Differences in the prevalence of AIV infections by sex and by age classes of Northern Pintail and Mallard ducks were detected, but the significance of these differences is undefined. In the 500 paired samples, molecular screening detected positive birds at a higher rate than viral isolation (χ2 = 8.35, p = 0.0035, df = 1); however, 20 AIV isolates were recovered from PCR-negative ducks. Further research is warranted to compare the two screening protocols’ potential for estimating true prevalence in wild birds. Our success during 2005 indicates Minto Flats will be a valuable study site for a longitudinal research project designed to gain further insight into the natural history, evolution, and ecology of AIV in wild birds.

Using Mean Infectious Dose of High-and Low-Pathogenicity Avian Influenza Viruses Originating from Wild Duck and Poultry as One Measure of Infectivity and Adaptation to Poultry

Abstract The mean infectious doses of selected avian influenza virus (AIV) isolates, determined in domestic poultry under experimental conditions, were shown to be both host-dependent and virus strain–dependent and could be considered one measure of the infectivity and adaptation to a specific host. As such, the mean infectious dose could serve as a quantitative predictor for which strains of AIV, given the right conditions, would be more likely transmitted to and maintained in a given species or subsequently cause an AI outbreak in the given species. The intranasal (IN) mean bird infectious doses (BID50) were determined for 11 high-pathogenicity AIV (HPAIV) isolates of turkey and chicken origin for white leghorn (WL) chickens, and for low-pathogenicity AIV (LPAIV) isolates of chicken (n  =  1) and wild mallards (n  =  2) for turkeys, and WL and white Plymouth rock (WPR) chickens, domestic ducks and geese, and Japanese quail. The BID50 for HPAIV isolates for WL chickens ranged from 101.2 to 104.7 mean embryo infectious dose (EID50) (median  =  102.9). For chicken-origin HPAIV isolates, the BID50 in WL chickens ranged from 101.2 to 103.0 EID50 (median  =  102.6), whereas for HPAIV isolates of turkey origin, the BID50 in WL chickens was higher, ranging from 102.8 to 104.7 EID50 (median  =  103.9). The BID50 of 104.7 was for a turkey-origin HPAIV virus that was not transmitted to chickens on the same farm, suggesting that, under the specific conditions present on that farm, there was insufficient infectivity, adaptation, or exposure to that virus population for sustained chicken transmission. Although the upper BID50 limit for predicting infectivity and sustainable transmissibility for a specific species is unknown, a BID50 < 104.7 was suggestive of such transmissibility. For the LPAIVs, there was a trend for domestic ducks and geese and Japanese quail to have the greatest susceptible and for WL chickens to be the most resistant, but turkeys were susceptible to two LPAIV tested when used at moderate challenge doses. This suggests domestic ducks and geese, turkeys, and Japanese quail could serve as bridging species for LPAIVs from wild waterfowl to chickens and other gallinaceous poultry. These data do provide support for the commonly held and intuitive belief that mixing of poultry species during rearing and in outdoor production systems is a major risk factor for interspecies transmission of AIVs and for the emergence of new AIV strains capable of causing AI outbreaks because these situations present a more diverse host population to circumvent the natural host dependency or host range of circulating viruses. Utilización de la dosis infecciosa media de virus de influenza aviar de alta o baja patogenicidad originados de aves domésticas o patos silvestres como una medida de infectividad y adaptación a las aves domésticas. Las dosis infecciosas medias de aislamientos seleccionados del virus de influenza aviar determinadas bajo condiciones experimentales en aves domésticas, demostraron ser dependientes del huésped y de la cepa del virus, pudiendo considerarse como una medida de la infectividad y adaptación a un huésped específico. Como tal, las dosis infectivas medias podrían servir para predecir cuantitativamente cuáles cepas del virus de influenza aviar, dadas las condiciones adecuadas, sería más propensa a ser transmitida y mantenida en una especie determinada o subsecuentemente causar un brote de influenza aviar es esa especie. En aves leghorn blancas, se calculó la dosis infecciosa media 50 por ave (por sus siglas en Inglés BID50) de 11 aislamientos de influenza aviar de alta patogenicidad originados en pollos y pavos posterior a la inoculación intranasal. A su vez, para aislamientos del virus de influenza aviar de baja patogenicidad originados en pollos y dos patos silvestres, se calculó el BID50 en pavos, aves leghorn blancas y plymouth rock blancas, patos domésticos, gansos y codornices Japonesas. El BID50 para los aislamientos de influenza aviar de alta patogenicidad (calculado en aves leghorn blancas) se presentó en el rango de 101.2 a 10 4.7 dosis infecciosa media de embrión 50 (DIE50) con un promedio de 102.9, mientras que para los aislamientos de influenza aviar de alta patogenicidad originados de pavos, el BID50 calculado en aves leghorn blancas fue mayor y en el rango de 102.8 a 104.7 DIE50 con un promedio de 103.9. El BID50 de 104.7 correspondió a un aislamiento de influenza aviar de alta patogenicidad originado en pavo que no se transmitió a pollos presentes en la misma granja, sugiriendo que bajo las condiciones específicas presentes en esa granja, hubo adaptación o en su defecto infectividad o exposición insuficiente a esa población viral para que se produjese la transmisión sostenida a los pollos. Aun cuando el límite superior de BID50 requerido para predecir infectividad y transmisibilidad sostenida se desconoce, un BID50 < 104.7 sugiere esa transmisibilidad. Para los virus de influenza aviar de baja patogenicidad, se observó una tendencia en los patos domésticos, gansos y en las codornices a una mayor susceptibilidad y en las aves leghorn blancas a ser mas resistentes. Los pavos fueron susceptibles a dos virus de influenza aviar de baja patogenicidad cuando se utilizaron dosis de desafío moderadas. Esto sugiere que los patos domésticos, gansos, pavos y las codornices Japonesas son especies que pueden servir como puente para virus de influenza aviar de baja patogenicidad provenientes de aves acuáticas hacia pollos y otras especies gallináceas domésticas. Estos datos proporcionan soporte para la creencia común e intuitiva de que mezclar especies de aves domésticas durante la crianza y en los sistemas de producción al aire libre es un factor de riesgo importante para la transmisión de virus de influenza aviar y para la emergencia de nuevas cepas del virus capaces de causar brotes de influenza aviar. Esto debido a que en estas situaciones se presenta una población de huéspedes más diversa que permite resolver las limitaciones del rango de huésped o dependencia al huésped de los virus circulantes. Abbreviations: AGP = agar gel precipitin; AI = avian influenza; BID50 = mean bird infectious dose; BLD50 = mean bird lethal dose; EID50 = mean chicken embryo infectious dose; HPAI = high pathogenicity avian influenza; HPAIV = high pathogenicity avian influenza virus; LPAI = low pathogenicity avian influenza; LPAIV = low pathogenicity avian influenza virus; LPM = live poultry market; Ro = reproductive ratio; SPF = specific pathogen free; WL = white leghorn; WPR = white Plymouth rock

Virus Replication in the Digestive Tract of Ducks Exposed by Aerosol to Type-A Influenza

Enteric infection and cloacal shedding of influenza virus was demonstrated in ducks exposed experimentally to an aerosol of an avirulent type-A influenza virus. The fluorescent-antibldy technique was used to identify sites of virus replication in the epithelial cells of the digestive tract and the bursa.

Characterization of Low-Pathogenicity H5N1 Avian Influenza Viruses from North America

ABSTRACT Wild-bird surveillance in North America for avian influenza (AI) viruses with a goal of early identification of the Asian H5N1 highly pathogenic AI virus has identified at least six low-pathogenicity H5N1 AI viruses between 2004 and 2006. The hemagglutinin (HA) and neuraminidase (NA) genes from all 6 H5N1 viruses and an additional 38 North American wild-bird-origin H5 subtype and 28 N1 subtype viruses were sequenced and compared with sequences available in GenBank by phylogenetic analysis. Both HA and NA were phylogenetically distinct from those for viruses from outside of North America and from those for viruses recovered from mammals. Four of the H5N1 AI viruses were characterized as low pathogenicity by standard in vivo pathotyping tests. One of the H5N1 viruses, A/MuteSwan/MI/451072-2/06, was shown to replicate to low titers in chickens, turkeys, and ducks. However, transmission of A/MuteSwan/MI/451072-2/06 was more efficient among ducks than among chickens or turkeys based on virus shed. The 50% chicken infectious dose for A/MuteSwan/MI/451072-2/06 and three other wild-waterfowl-origin H5 viruses were also determined and were between 105.3 and 107.5 50% egg infective doses. Finally, seven H5 viruses representing different phylogenetic clades were evaluated for their antigenic relatedness by hemagglutination inhibition assay, showing that the antigenic relatedness was largely associated with geographic origin. Overall, the data support the conclusion that North American H5 wild-bird-origin AI viruses are low-pathogenicity wild-bird-adapted viruses and are antigenically and genetically distinct from the highly pathogenic Asian H5N1 virus lineage.

Evolution of H5 subtype avian influenza A viruses in North America

The phylogenetic relationships of the hemagglutinin (HA) and non-structural (NS) genes from avian influenza (AI) H5 subtype viruses of North American origin are presented. Analysis of the HA genes of several previously uncharacterized isolates from waterfowl and turkeys provided clear evidence of significant sequence variation and existence of multiple virus clades or sub-lineages, maintained in migratory waterfowl. Phylogenetic analysis of NS gene sequences further demonstrated multiple sub-lineages and also demonstrated re-assortment of two NS alleles in wild duck populations. Based on currently available HA1 gene sequences, at least four clades exist with waterfowl isolates included in three of the four groups. The most genetically unstable of these sub-lineages is composed of recent poultry isolates from the outbreak of AI in Central Mexico. This group of viruses, which replicated unabated in chickens for at least 16 months, exhibited an increased rate of mutation in both the HA and NS gene. Comparison of the HA1 sequence data for all available North American H5 subtype viruses demonstrated minimal variation both in and around the amino acids predicted to be involved in the HA receptor binding site. The sequences also revealed that migratory waterfowl, live poultry market chicken, and turkey isolates uniformly lack a glycosylation site at amino acid 236 in the HA protein which is present in commercial chicken isolates.

Replication of a Waterfowl-Origin Influenza Virus in the Kidney and Intestine of Chickens

Intravenous inoculation of chickens with a waterfowl-origin type A influenza virus resulted in high titers of virus in kidney tissues and viral nucleoprotein in renal tubular epithelial cells and in intestinal mucosal epithelial cells. Virus titers in kidneys of four of eight clinically normal chickens sampled on days 3 and 5 postinoculation (PI), one dead chicken on day 3 PI, and one dead chicken on day 7 PI exceeded 10(6) mean embryo infectious dose per gram of tissue. Using immunofluorescent and immunoperoxidase staining, viral nucleoprotein was identified in the cytoplasm and nucleus of tubular epithelial cells in kidneys and in nucleus of mucosal epithelial cells lining villi in the lower small intestine. Based on the low intravenous pathogenicity index for this virus (0.3) along with the high virus titers in kidney tissues and localization of viral antigen in kidney important site for replication of avian influenza (AI) virus of low pathogenicity. Recovery of type A influenza viruses from cloacal swabs could result from viral replication in kidneys as well as in the lower intestine and/or the bursa of Fabricius.

Examining the hemagglutinin subtype diversity among wild duck-origin influenza A viruses using ethanol-fixed cloacal swabs and a novel RT-PCR method

This study presents an interconnected approach for circumventing two inherent limitations associated with studies defining the natural history of influenza A viruses in wild birds. The first limiting factor is the ability to maintain a cold chain from specimen collection to the laboratory when study sites are in more remote locations. The second limiting factor is the ability to identify all influenza A virus HA subtypes present in an original sample. We report a novel method for molecular subtyping of avian influenza A virus hemagglutinin genes using degenerate primers designed to amplify all known hemagglutinin subtypes. It was shown previously that templates larger than 200 bp were not consistently amplifiable from ethanol-fixed cloacal swabs. For this study, new primer sets were designed within these constraints. This method was used to perform subtyping RT-PCR on 191 influenza RNA-positive ethanol-fixed cloacal swabs obtained from 880 wild ducks in central Alaska in 2005. Seven different co-circulating hemagglutinin subtypes were identified in this study set, including H1, H3, H4, H5, H6, H8, and H12. In addition, 16% of original cloacal samples showed evidence of mixed infection, with samples yielding from two-to-five different hemagglutinin subtypes. This study further demonstrates the complex ecobiology of avian influenza A viruses in wild birds.