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Isolation of H5 Avian Influenza Viruses from Waterfowl in the Upper MidwestRegion of the United StatesAuthor(s): Mohamed E. El Zowalaty, Martha Abin, Yogesh Chander, Patrick T. Redig, and Sagar M.GoyalSource: Avian Diseases, 55(2):259-262. 2011.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/9477-072110-Reg.1URL: http://www.bioone.org/doi/full/10.1637/9477-072110-Reg.1

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Isolation of H5 Avian Influenza Viruses from Waterfowl in theUpper Midwest Region of the United States

Mohamed E. El Zowalaty,AB Martha Abin,A Yogesh Chander,A Patrick T. Redig,A and Sagar M. GoyalAC

ADepartment of Veterinary Population Medicine and Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota,Saint Paul, MN 55108

BDepartment of Microbiology and Immunology, Faculty of Pharmacy, University of Zagazig, Zagazig, 44519, Egypt

Received 23 July 2010; Accepted and published ahead of print 7 February 2011

SUMMARY. In recent years, the H5N1 subtype of avian influenza virus (AIV) has become an important zoonotic pathogen.The surveillance of AIV in its natural host, the waterfowl, is crucial to monitoring and controlling the disease in poultry and otherspecies. In this study, we report on the isolation of H5 AIV from cloacal swabs of waterfowl captured in Minnesota and SouthDakota. We screened a total of 7260 cloacal samples from waterfowl using matrix gene–directed, real-time reverse transcription–(rRT-PCR) and H5-specific rRT-PCR and found 148 samples to be positive for the H5 subtype. On inoculation of 71 of thesesamples in embryonated chicken eggs, 25 samples yielded H5 AIV. On subtyping with N-specific primers, we detected a mixture ofsubtypes in 15 isolates. Molecular pathotyping confirmed the isolated H5 subtypes to be low pathogenicity avian influenza.Continuation of AIV surveillance programs should help in understanding the epidemiology and ecology of AIV.

RESUMEN. Aislamiento de virus de la influenza aviar H5 de aves acuaticas de la parte norte de la zona medio oeste de losEstados Unidos.

En los ultimos anos, el subtipo H5N1 del virus de la influenza aviar se ha convertido en un patogeno zoonotico importante. Lavigilancia del virus de influenza aviar en su huesped natural, las aves acuaticas, es crucial para llevar a cabo el seguimiento y elcontrol de la enfermedad en la avicultura y en otras especies. En este estudio, se reporta el aislamiento del virus de influenza aviarH5 de hisopos cloacales de aves capturadas en Minnesota y en Dakota del Sur. Se analizaron un total de 7,260 muestras cloacales deaves acuaticas mediante la tecnica de la transcripcion reversa y PCR en tiempo real dirigida hacia el gene de la matriz y tambien porla tecnica especıfica para el gene H5. Se encontraron 148 muestras positivas para el subtipo H5. Mediante la inoculacion en huevosembrionados de pollo de 71 de estas muestras, 25 muestras mostraron la presencia del virus de la influenza aviar H5. Mediante lasubtipificacion utilizando iniciadores N-especıficos, se detecto una mezcla de subtipos en 15 aislamientos. La patotipificacionmolecular confirmo que los aislamientos del subtipo H5 eran de baja patogenicidad. La continuacion de los programas de vigilanciade los virus de influenza aviar deben ayudar en la comprension de la epidemiologıa y de la ecologıa del virus de influenza aviar.

Key words: avian influenza, H5 subtype, waterfowl, Minnesota, South Dakota

Abbreviations: AIV 5 avian influenza virus; ECE 5 embryonated chicken eggs; HPAI 5 high pathogenicity avian influenza;LPAI 5 low pathogenicity avian influenza; rRT-PCR 5 real-time reverse transcription–PCR; RT-PCR 5 reverse transcription–PCR; SPF 5 specific pathogen free

To date 16 HA and nine NA subtypes of avian influenza virus(AIV) have been identified, and almost all of them have beendetected in waterfowl (16,21,26) in various combinations; waterfowlare regarded as the natural reservoir for AIV. Based on pathogenicity,there are two groups of AIV: low pathogenic avian influenza (LPAI),which causes mild or undetectable disease, and highly pathogenicavian influenza (HPAI), which causes severe disease and highmortality in domestic poultry within a short period of time (4).

Of the 16 HA subtypes, H5 and H7 subtypes represent a potentialto be HPAI and are thus notifiable to the Office International desEpizooties (15). The virulent subtypes can arise from avirulent strainsthrough the loss of specific sequences, such as the glycosylation sites, orby gaining multiple basic amino acid sequences at the HA cleavage site(10). Since its emergence in Asia in 1996, HPAI H5N1 has crossed thespecies boundary and now infects other host species, includinghumans, in whom it has a high case fatality rate (1).

Since its global spread, H5 AIV in general, and H5N1 and H5N2in particular, have gained attention among public health officials as aresult of their potential risk as HPAI and because of their ability tocause disease in poultry and mammals (6,7,13,14). Notifiable AIVhas been reported from several countries, either as sporadic cases oras outbreaks in poultry. Thus, AIV surveillance in waterfowl is

necessary to determine the current situation related to circulatingAIV subtypes and their pathogenic potential (3). We conducted thisstudy to determine the prevalence of H5 AIV in cloacal samplesfrom waterfowl in the upper Midwest region of the United States.

MATERIALS AND METHODS

Source of samples and sample processing. From June to September of2008, cloacal swabs from 7260 migrating waterfowl were collected. Thebirds were trapped and sampled in the states of Minnesota, North Dakota,and South Dakota from 29 different locations using night lighting, rocketnetting, and hunter harvesting. The swabs were placed in brain heartinfusion broth containing penicillin (10,000 units/ml), streptomycin(10,000 mg/ml), gentamicin (5000 mg/ml), and amphotericin B (50,000mg/ml). The samples were transported to the laboratory under refrigerationconditions and stored at 280 C until they were processed.

Experimental plan. Pools of cloacal samples (five samples per pool)were tested for AIV in a matrix gene–based real-time reversetranscription–PCR (rRT-PCR). Individual samples from positive poolswere retested by rRT-PCR, and those found to be positive were testedfor H5 subtype using H5-specific rRT-PCR (19). The H5-positivesamples were inoculated in 9-day-old specific-pathogen-free (SPF)embryonated chicken eggs (ECE) for virus isolation. Viral isolates weresubtyped by molecular methods and were subjected to molecularpathotyping to determine their pathogenicity. Details of the methods aregiven below.CCorresponding author. E-mail: goyal001@umn.edu

AVIAN DISEASES 55:259–262, 2011

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RNA extraction from field samples and allantoic fluid. RNA fromindividual cloacal samples and their pools was extracted using anAmbion MagMAXTM RNA isolation kit (Ambion, Inc., Austin, TX).RNA from infected allantoic fluids was extracted using TRIzolH(Invitrogen, Carlsbad, CA).

Virus isolation. Of the 148 cloacal samples positive for H5 by rRT-PCR, 71 were inoculated into 9-day-old SPF ECE by the allantoic routeusing two eggs per sample (200 ml of sample per egg). Samples werepassed for three blind passages in ECE. The eggs were incubated at 37 Cfor 4 days and were candled daily to determine embryo viability. At theend of 4 days of incubation or upon embryo death, the eggs were chilledat 4 C for 24 hr and the allantoic fluids were harvested aseptically andtested for hemagglutination activity using turkey erythrocytes (0.5% in0.01 M phosphate-buffered saline, pH 7.2).

Subtyping procedure. For subtyping, RNA was extracted from HA-positive allantoic fluids (19). For amplification of HA and NA, themethods described by Hoffmann et al. (9) and Chander et al. (2) wereused. PCR products were purified and submitted for sequencing to theBiomedical Genomic Center, University of Minnesota (Saint Paul, MN)in both directions using the same mixture of primers as was used foramplification. The sequence data were aligned with the existinginfluenza database using the BLAST search tool, available online(www.ncbi.nlm.nih.gov), to determine the subtype.

Pathotyping of H5 isolates. To determine the pathogenic potentialof H5 isolates, the HA cleavage site was amplified by the RT-PCRmethod, as described previously (24). Bands corresponding to HAcleavage site of 249 bp were excised and purified using the QIAquickGel Extraction kit (Qiagen, Valencia, CA). The purified DNAfragments were sequenced. The nucleotide sequences were used todetermine the amino acid sequence at the HA cleavage site usingSequencher 4.9 software, available at the Minnesota SupercomputingInstitute, University of Minnesota (Saint Paul, MN).

RESULTS

The main objective of this study is to present data on H5 AIV toindicate that they are fairly widespread in the waterfowl populationand should not be ignored. Isolation of other non-H5 AIVserotypes from rRT-PCR–negative samples within the 7260samples has been reported elsewhere (4). Of the 7260 cloacalsamples tested, 487 pools were positive, out of which 890

individual samples were positive for AIV by rRT-PCR. Whenthese 890 samples were tested with H5-specific rRT-PCR, 148(2%) were H5 positive. A majority of H5-positive samples camefrom the following species: 87 mallards (Anas platyrhynchos), 38northern pintail (Anas acuta), and seven each of American green-winged teal (Anas carolinensis) and blue-winged teal (Anas discors;Table 1). Adequate sample volume was available for only 71 of148 H5-positive samples. These 71 samples were inoculated inECE, followed by two additional blind passages. The resultingallantoic fluids were tested for HA activity, and 25 were HApositive and were confirmed to be AIV by rRT-PCR. The 148PCR-positive samples out of a total of 7260 translates to an H5prevalence rate of 2.04%. On subtyping with N-specific primers,eight samples were found to be H5N2, while 15 samples had amixture of H5N1 and H5N2, which was confirmed by sequencingand BLAST analysis. The remaining two samples were subtypedwith Hoffmann primers for H (9); one had a mixture of H5N1and H1N1 and the other one had H5N8 and H4N8 (Table 1),which was confirmed by nucleotide sequencing and BLASTanalysis. These 25 samples came from the following species: 13northern pintail (Anas acuta), seven mallards (Anas platyrhynchos),four from American green-winged teal (Anas carolinensis), and onefrom blue-winged teal (Anas discors). To determine theirpathogenic potential, the HA cleavage site of these isolates wassequenced. All isolates were LPAI with an amino acid sequence ofRETR at the HA cleavage site (24).

DISCUSSION

Wild aquatic birds are the major reservoirs of influenza A virusesand have directly or indirectly been recognized as the main source ofAIVs infecting avian and non-avian species (21). Because of itspotential risk to domestic poultry, surveillance of H5 AIV in itsnatural host is important epidemiologically (27). It is generallyaccepted that H5 LPAI can mutate to HPAI after transmission toother avian hosts species (12). Surveillance of waterfowl should helpin understanding the evolution and ecology of AIV, which will behelpful in combating this virus (21).

Table 1. Detection of H5 subtype avian influenza viruses (AIVs) in rRT-PCRA-positive cloacal samples of waterfowl.

Bird species No. of cloacal samples testedB No. positive for HAC SubtypesD

Mallard (Anas platyrhynchos) 87 7 H5N2 (n 5 3)H5N1 and H5N2 (n 5 3)E

H5N1 and H1N1 (n 5 1)E

Northern pintail (Anas acuta) 38 13 H5N2 (n 5 3)H5N1 and H5N2 (n 5 10)E

American green-winged teal (Anas carolinensis) 7 4 H5N2 (n 5 2)H5N1 and H5N2 (n 5 2)E

Blue-winged teal (Anas discors) 7 1 H5N8 and H4N8 (n 5 1)E

Ring-necked duck (Aythya collaris) 2 0 0Wood duck (Aix sponsa) 2 0 0Pied-billed grebe (Podilymbus podiceps) 1 0 0Canada goose (Branta mericana) 1 0 0Red-headed duck (Aythya americana) 1 0 0Unidentified birds 2 0 0Total 148 25 25

AAIV rRT-PCR, according to Spackman et al. (2002).BNo. of individual positive samples tested by rRT-PCR and virus isolation within each species.CHemagglutination results after egg inoculation of H5 AIV rRT-PCR–positive samples.DNo. that could be subtyped using primers by Hoffman et al. (9) and by Chander et al. (2).EMixed infections with two subtypes of AIV.

260 M. E. El Zowalaty et al.

Outbreaks of Asian-lineage H5N1 HPAI in several parts of theworld since 1996 have been associated with wild bird transmission insome cases. Hence, waterfowl surveillance programs involving H5AIV have been initiated in the United States (20). Detection ofH5N1 and H5N2 in our study reinforces the continuous need for anintensive surveillance program. Although the detected H5N1 andH5N2 isolates in this study were of low pathogenicity, the potentialexists for them to become HPAI (23). Further characterization ofwaterfowl-origin H5 AIV should lead to a better understanding ofthe ecology of H5 AIV in waterfowl as well as of the likelihood thatthey will cross species barriers and infect other hosts (20).

Although H5 was thought to be one of the less common HAsubtypes isolated from waterfowl in North America (11,22),increased AIV surveillance has resulted in frequent isolation of thissubtype (20). It has also been determined that differences in H5prevalence could be due to year-to-year variation. There have beenfour large outbreaks of LPAI in Minnesota turkeys since the mid-1970s (7). In the United States, a large outbreak is defined asinvolving more than 100 flocks or one million birds. The sources ofall four outbreaks in Minnesota were considered to be wild birds.The economic losses due to these four outbreaks were estimated tobe more than US$21 million (7,8). In today’s dollars, this amountwould even be larger.

Mixed infections were detected in 17 of 25 AIV-positive samples.This is not surprising because mixed AIV infections (more than oneHA and/or NA subtypes) in cloacal samples of waterfowl havepreviously been reported (17,25). In one study, 18 of 29 isolatescontained more than one HA and/or NA subtype (18). Mixedinfections have also been reported among Minnesota ducks (8). Inanother report, a high rate of mixed infection was noted, and two tofive different HA subtypes were isolated from 16% of the samples(25). Characterization of mixed AIV infections is important to betterunderstand the contribution of genetic diversity toward ecology andepidemiology of AIV (5,25).

Only 25 of 71 samples positive by rRT-PCR yielded virus oninoculation in ECE. The remaining 46 samples were virus isolationnegative. This is probably due to the inability of all AIV strains togrow to detectable titers within three ECE passages (19). We believethat additional blind passages of such samples in ECE may improvethe rate of virus isolation. Another explanation is that the virus maybe dead, in which case it could be detected by rRT-PCR but not byvirus isolation.

In summary, we have reported on the occurrence of H5N1 andH5N2 AIV subtypes of wild-bird origin in the upper Midwestportion of the United States. In addition, surveillance for other AIVsubtypes will certainly contribute to understanding the epidemiologyof AIV. This emphasizes the need for continuation of AIVsurveillance programs throughout different parts of the UnitedStates so that we can gain a better understanding of the ecology ofAIV and in order to improve the preparedness measures for theprotection of the poultry industry.

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ACKNOWLEDGMENTS

This work has been funded in whole or in part with federal fundsfrom the National Institute of Allergy and Infectious Diseases, NationalInstitutes of Health, Department of Health and Human Services, underContract No. HHSN266200700007C. Its contents are solely theresponsibility of the authors and do not necessarily represent the officialviews of the National Institutes of Health. We thank the EgyptianCultural and Educational Bureau in Washington, DC, ZagazigUniversity, and the Egyptian Ministry of Higher Education and Statefor Scientific Research for financial support of M. Ezzat El Zowalaty.

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