Serologic tests.

Antisera specific for the isolated HA and NA antigens of the reference strains of influenza A viruses were prepared in goats (28). Monoclonal antibodies to A/chicken/Pennsylvania/1370/83 (H5N2) were prepared as described previously (13). HA titrations and HI tests were performed in microtiter plates with sera that had been treated with a receptor-destroying enzyme (15). NA titrations and NA inhibition tests were done according to the procedure of Aymard-Henry et al. (4).

Infection studies of poultry.

The viruses used were grown in embryonated chicken eggs, and aliquots of the virus were stored at −70°C (see tables and Fig. ​Fig.22 for details). The 50% egg infectious doses (EID₅₀) were determined from samples of groups of three infected eggs at days 3 and 5 after infection.

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FIG. 2.

Changes in the weights of mice infected with goose H5N1 influenza viruses. BALB/c mice were infected with Gs/HK/437-4/99 (), Gs/HK/437-6/99 (•), Gs/HK/437-8/99 (), Gs/HK/437-10/99 (•), Gs/HK/485-3/00 (○), and Gs/HK/485-5/00 (□). Untreated animals () received PBS. The weights of the mice were measured on days 0, 3, 5, 7, 9, 11, 14, and 16 after infection. Each value is the percent change in the initial mean starting weight on day 0. The standard errors (SE) are given.

Chinese white geese, 3 to 4 weeks old, were inoculated with virus in a volume of 1.0 ml via tracheal and oral routes and via the nares and eyes (see Table ​Table33 for doses). Virus infectivity titers were determined from samples of brain, heart, lung, liver, spleen, kidney, and intestine from animals that died of infection or that were very sick and were euthanized. In addition, tracheal and cloacal samples were collected at different intervals, and influenza viruses isolated from these samples were titrated in embryonated eggs.

The pathogenicities of the viruses in chickens were determined as described previously (18). Three-week-old specific-pathogen-free chickens received inoculations with virus intravenously (injection volume, 0.2 ml) or tracheally or orally and into the nares and eyes in a total volume of 1.0 ml. Blood samples were collected from chickens 14 and 21 days after infection for serologic analysis. Tracheal and cloacal swabs were collected and analyzed for virus in chicken embryos. The 50% chicken infectious dose (dose of virus infecting 50% of inoculated chickens; CID₅₀) was determined by infecting groups of four chickens with 10-fold dilutions of virus grown in chicken embryos. Chickens were swabbed tracheally and cloacally on days 3 and 5, and virus was detected by replication in 10-day-old chicken embryos. The 50% chicken lethal dose (CLD₅₀) was determined from the number of deaths in the groups of infected chickens 10 days after infection.

Groups of three young adult quail (Coturnix coturnix) were infected orally, intranasally, and orbitally with 10^6.5 EID₅₀ of virus in a volume of 0.5 ml. Tracheal and cloacal swabs were obtained, and the amounts of virus present in samples from infected quail were measured as described previously (9). The methods of determining the 50% quail infectious dose (QID₅₀) and the 50% quail lethal dose (QLD₅₀) were the same as those used in determining the CID₅₀ and CLD₅₀.

Young Pekin white ducks (2 months old) were inoculated with approximately 10^6.5 EID₅₀ of virus in a total volume of 1.0 ml. The sites of inoculation were the trachea, the nares, eyes, and oral cavity (22). Tracheal and cloacal swabs were collected at different intervals, and influenza viruses within these samples were titrated in embryonated eggs.

All studies were carried out in U.S. Department of Agriculture-approved BL3⁺ facilities with animals that were free of influenza virus that could be detected by serologic or virologic assays. Because all experiments were done in a BL3⁺ containment facility, the total number of birds used in the experiments was limited by the amount of space in the facility (see Tables ​Tables33 to ​to77 for details). All poultry were observed for signs of disease, and food and water intake was monitored; the research complied with all relevant federal guidelines and institutional policies related to animal care.

Transmission of H5N1 virus from geese to chickens or to quail.

Three- to 4-week-old Chinese white geese were housed in 0.4-m² wire mesh cages and fed ad libitum. The birds were inoculated with 10^5.75 EID₅₀ of A/goose/Hong Kong/437-4/99 (A/Gs/HK/437-4/99; H5N1) virus in a total volume of 1.0 ml by the tracheal and oral routes and via the eyes and nares. The infected birds were put into contact with three geese of the same age. Uninfected quail or specific-pathogen-free white leghorn chickens were placed in cages below and beside the cage containing the infected geese; the distance between the cages was approximately 30 cm. The dropping tray was removed from the goose cage to allow direct contact between the birds below and the feces of infected birds. Air in the containment cabinets flowed from the bottom through the top.

Infection studies of mice.

Female BALB/c mice (weight, 18 to 20 g; The Jackson Laboratory, Bar Harbor, Maine) were used. After the mice were anesthetized by inhaling isoflurane, each was intranasally inoculated with 10^6.5 or 10^6.75 EID₅₀ of virus in a volume of 0.1 ml. Eight to 10 mice per group were observed daily for signs of infection and survival until 16 days after infection. The mice were weighed on days 0, 3, 5, 7, 9, 11, 14, and 16 after infection. Three mice from each experimental group were exsanguinated on days 3 and 7, and their lungs and brains were removed. The organs were ground and homogenized in 1 ml of cold phosphate-buffered saline (PBS), solid debris was pelleted by centrifugation at 2,000 × g for 10 min, and the tissues were used to determine the EID₅₀ in 10-day-old embryonated chicken eggs. Virus titers in mouse lungs and brain are given in units of log₁₀ EID₅₀ per 0.1 ml ± standard error (SE). The limit of virus detection was 0.1 log₁₀ EID₅₀ per 0.1 ml. For dose-response experiments, BALB/c mice were infected with 10^7.2, 10^6.5, 10^5.5, 10^4.5, or 10^3.5 EID₅₀ of A/Gs/HK/437-10/99 or A/Gs/HK/485-5/00 isolates in a volume of 0.1 ml.

Gene sequencing and analysis.

Viral gene sequencing and analysis were performed as previously described (10). In brief, we used the RNeasy Mini Kit (Qiagen, Inc., Valencia, Calif.) to extract RNA from viruses in infected allantoic fluid. Reverse transcription-PCR (RT-PCR) was performed with specific primers for each gene segment (primer sequences are available upon request). PCR products were purified with the QIAquick PCR purification kit (Qiagen, Inc.) and sequenced with synthetic oligonucleotides. Reactions were performed with rhodamine DyeTerminator cycle sequencing ready reaction kits and AmpliTaq DNA polymerase FS (Perkin-Elmer/Applied Biosystems, Inc.). Samples were subjected to electrophoresis and analyzed on model 377 DNA sequencers (Perkin-Elmer/Applied Biosystems, Inc.).

The sequence data were edited by using the Wisconsin sequence analysis package, version 10.0 (Genetics Computer Group, Madison, Wis.). Phylogenetic analyses were performed with PAUP (phylogenetic analysis using parsimony), version 4.0 (David Swofford, Illinois Natural History Survey, Champaign, Ill.).

Antigenic characterization of H5N1 goose influenza virus isolates.

To determine the antigenic subtype of the influenza viruses from geese and their relationship to other influenza viruses, the six goose influenza virus isolates were examined with a panel of reference sera to the 15 HA and 9 NA subtypes and with a panel of monoclonal antibodies to A/chicken/Pennsylvania/1370/83 (H5N2). Antigenic analysis with hyperimmune goat antisera to A/tern/South Africa/61 (H5N3) and monoclonal antibodies to A/chicken/Pennsylvania/1370/83 (H5) showed that the viruses are of the H5 subtype and are closely related, if not identical, to H5N1/97 (H5N1) (Table ​(Table1).1). One goose isolate (Gs/HK/437-6/99) was antigenically distinguishable from the rest when analyzed with hyperimmune sera and with monoclonal antibody CP-46. This result indicates some antigenic heterogeneity between Gs/HK/437-6/99 and the other isolates. NA inhibition tests showed that the NA of each isolate belongs to the N1 subtype.

Genetic analysis of goose H5N1 viruses.

To determine the extent of genetic relatedness between the Go/Gd-like isolates collected in 2000 and those collected in 1996 and 1999 (7, 30), one virus (Gs/HK/485-3/00) was completely sequenced and compared with three representative viruses that were partially sequenced (Table ​(Table2).2). The analyzed gene segments from the two H5N1 Go/Gd-like isolates collected in 2000 were 99.6 to 100% homologous with each other. In addition, the gene segments of these H5N1 isolates were highly related to those of the isolates collected in 1996 and 1999. The PB2 and NA gene segments of the February 2000 isolates were 97.6 and 97.9% homologous with those of goose/Guangdong/1/96 and 99.2% homologous with those of the 1999 isolates (Table ​(Table22).

Sequence analysis showed a limited number of amino acid changes scattered throughout the HA molecules. One change in the 2000 Gs/Gd isolates with potential functional relevance was the arginine-to-glycine substitution at residue −6 of HA1, resulting in shortening the series of basic amino acids associated with the HA of highly pathogenic avian influenza viruses. This substitution reduced the number of basic amino acids at the cleavage site from six to five. Sequence analysis showed no changes in the length of the stalk of the NA molecules.

Phylogenetic analysis of each gene segment of the 2000 H5N1 goose isolates revealed genetic relationships similar to those previously reported for the 1996 and 1999 goose H5N1 isolates (7, 30). Phylogenetic analysis of the HA1 molecules showed that each goose H5N1 isolate from 1996, 1999, and 2000 is part of a separate sublineage (Fig. ​(Fig.1),1), whereas the HAs of H5N1 viruses isolated from poultry and humans in 1997 form a separate sublineage. Phylogenetic analysis of the seven other gene segments of the goose H5N1 isolates collected in 2000 showed a high degree of relatedness to goose H5N1 isolates from 1996 and 1999 but showed a marked difference from H5N1 isolates from humans and chickens in 1997 (results not shown).

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FIG. 1.

Phylogenetic analysis of the HA1 genes of H5 influenza A viruses. The tree was generated with PAUP by using a maximum-parsimony algorithm. Nucleotides 77 through 1037 (961 bp) of the HA gene were analyzed. The tree is rooted to A/Japan/305/57 (H2N2). Only Eurasian lineages are shown. The lengths of the horizontal lines are proportional to the minimum numbers of nucleotide differences required to join nodes. Vertical lines are for spacing branches and labels. Abbreviations: Ck, chicken; Dk, duck; Env, environment; Gs, goose; Ty, turkey; GD, Guangdong; HK, Hong Kong.

Transmission and pathogenicity of goose H5N1 influenza viruses in geese.

In the field, A/goose/Guangdong/1/96 (H5N1) influenza virus reportedly killed 40% of infected geese (26). In contrast, no deaths occurred and no signs of disease were present among geese in the poultry market that contained H5N1 Go/Gd-like viruses in the present studies. These H5N1 viruses were isolated from goose holding pens or from cloacal samples at the port of entry into Hong Kong after shipment by boat from southern China.

To determine the pathogenicity and transmissibility of these H5N1 viruses, geese were inoculated with four isolates collected in 1999 and two from 2000. Four groups of geese were infected with one each of the four isolates and put into contact with uninfected geese. Three of the four H5N1 isolates collected in 1999 killed the inoculated animals, and one of the 2000 isolates also killed one of three inoculated geese (Table ​(Table3).3). The birds that died initially showed only mild signs of disease; some of these birds experienced weight loss, lethargy, and diarrhea. Virus was detected in tracheal and cloacal samples from all infected geese, and some surviving animals continued to shed virus for 12 days (data not shown). Titration of the organs from a dead goose (Gs/HK/485-5/00) revealed virus in the organs examined, the highest titers being in the liver (5.5 log₁₀ EID₅₀), kidney (4.50 log₁₀ EID₅₀), and lung (4.25 log₁₀ EID₅₀); lower titers were found in the heart (3.5 log₁₀ EID₅₀), brain (2.75 log₁₀ EID₅₀), and spleen (2.50 log₁₀ EID₅₀).

Each H5N1 isolate collected in 1999 was transmitted to geese that were in contact with the experimentally infected birds, and one goose died after being infected with the Gs/HK/437-4/99 isolate through contact. The other three isolates were detected in the trachea and cloaca for up to 5 days. These animals showed no overt signs of disease. In later transmission experiments, we used the single isolate that caused the death of one goose after it was infected through contact (i.e., Gs/HK/437-4/99) (data not shown). In these experiments, none of the four birds in contact with the infected birds, though infected, showed signs of disease or died of influenza infection. Despite the small number of geese that we were able to test, the overall findings indicate that high doses of virus can kill geese but that, when viruses are transmitted to geese by contact, the mortality rate is relatively low.

Replication and transmission of A/Gs/HK/99 (H5N1) influenza viruses in ducks.

Because domestic ducks are considered a possible intermediate host between wild aquatic ducks and other domestic poultry in the natural transmission of influenza viruses, we studied the ability of domestic ducks to support the replication of the goose H5N1 viruses. Two H5N1 isolates collected in 1999 were used to inoculate Pekin white ducks, which were subsequently put into contact with uninfected ducks (Table ​(Table4).4). Each H5N1 isolate replicated in the four inoculated animals in each group and were detected in the cloaca on days 3 or 5; the virus titers ranged from <2.0 to >5.0 log₁₀ EID₅₀ per 0.1 ml. Viruses were also detected in the tracheas of most birds; virus titers were from <2.0 to >5.5 log₁₀ EID₅₀ per 0.1 ml. Detection of H5N1 viruses in ducks that were in contact with the experimentally infected ducks varied; because the number of birds used in the experiment was small, we can only conclude that the viruses can be transmitted to ducks through contact and can be detected in the tracheas and the cloacae of ducks infected through contact. None of the ducks showed signs of disease, and they consumed normal amounts of food.

Pathogenicity of goose H5N1 influenza viruses in chickens.

The intravenous injection of virus-containing allantoic fluid into chickens is the U.S. Department of Agriculture’s approved method of determining the pathogenicity of influenza viruses in chickens. If the viruses are highly pathogenic, they will kill six of eight inoculated birds in 10 days (18). In addition, the amino acid sequence at the connecting peptide region of HA₀ is expected to possess a series of basic amino acids.

After intravenous injection into chickens, each of the six goose isolates collected in 1999 or 2000 caused disease signs typical of those seen in chickens infected with highly pathogenic influenza viruses, e.g., swollen heads and blue combs, and killed seven or all eight of the infected birds (Table ​(Table5).5). Oral or nasal inoculation of chickens with the 1999 goose isolates produced disease signs typical of those seen in chickens infected with highly pathogenic avian influenza viruses, but these routes of inoculation resulted in the deaths of only one or two of the five birds (Table ​(Table5).5). In contrast the 2000 isolates killed three-fourths of chickens when inoculated by the oral or nasal route. Thus, our findings indicate that both the 1999 and 2000 goose H5N1 isolates are highly pathogenic in chickens and that each virus has a series of basic amino acids adjacent to the HA cleavage site (Table ​(Table55).

Pathogenicity of goose H5N1 viruses in quail.

Because quail were the source of the A/quail/Hong Kong/G1/97 (H9N2) virus, which may have been the donor of genes encoding internal proteins for the H5N1/97-like viruses, we examined the ability of quail to support the replication of the goose H5N1 viruses, the reported donors of the HA gene for the H5N1/97-like viruses (30). Each goose H5N1 virus was highly pathogenic in quail that had been inoculated orally and intranasally (Table ​(Table5).5). The birds became lethargic by the third day and died between days 3 and 7 after inoculation. Titration of the organs of quail that died on days 3 to 5 revealed virus in all organs examined, the highest titers being detected in the brains (4.3 to 7.5 log₁₀ EID₅₀) and pancreases (0 to >5.0 log₁₀ EID₅₀), with lower titers in the lungs (3.55 to 4.5 log₁₀ EID₅₀) and liver (<1 to 2.8 log₁₀ EID₅₀). Thus each of the goose H5N1 viruses was highly pathogenic in quail.

Transmission of A/Gs/HK/437-4/1999 (H5N1) from geese to chickens and to quail.

Given the role of the goose in the genesis of the H5N1 virus in 1997, we determined whether H5N1 influenza viruses isolated from geese could spread from geese to other domestic poultry, i.e., chickens and quail. Chinese white geese were infected with the representative Gs/HK/437-4/99 isolate and put into the same cages as the uninfected birds. Transmission by contact with feces was also studied by housing quail or chickens in cages below the geese. Aerosol transmission of virus was studied by placing quail or chickens in cages that were beside the cages of infected geese at a distance of approximately 0.3 m.

Infection of geese with Gs/HK/437-4/99 resulted in the deaths of more than 50% of the birds (Table ​(Table6),6), but none of the geese that were in contact, with the infected ones died despite acquiring infection through heavy fecal contamination of the cages and water troughs. In contrast, all four chickens that had contact with goose feces became infected and two died after they showed signs typical of highly pathogenic influenza infection. Tracheal and cloacal samples collected on days 3, 5, 7, 9, and 14 contained virus (details not shown); virus was isolated more consistently from cloacal samples than from tracheal samples. In contrast to the results for fecal transmission to chickens, there was no evidence of aerosol transmission of virus to chickens. Studies of transmission of Gs/HK/437-4/99 (H5N1) to quail revealed that all birds (three of three) that were in contact with goose feces and two of the four birds with exposure to aerosol spread died after they had displayed signs typical of highly pathogenic influenza infection. In the quail studies, viruses were isolated more consistently from tracheal samples than from cloacal samples (results not shown). Thus, our findings indicate that quail were infected by aerosol exposure to geese infected with Gs/HK/437-4/99, whereas chickens were not.

Dose of A/Gs/HK/437-4/99 (H5N1) required to infect quail and chickens.

The above findings indicating that a representative H5N1 isolate (Gs/HK/437-4/99) can be transmitted by the aerosol route to quail but not to chickens raise the question of whether quail are more susceptible than chickens to infection with this virus. To answer this question, we determined the QID₅₀ and QLD₅₀ in quail and the CID₅₀ and CLD₅₀ in chickens of the Gs/HK/437-4/99 (H5N1) virus. The QID₅₀ and QLD₅₀ required 1.7 and 2.5 log₁₀ EID₅₀ of virus, respectively (average of two independent experiments), while the CID₅₀ and CLD₅₀ required 3.8 and 4.0 log₁₀ EID₅₀ of virus, respectively. Therefore, quail are more susceptible to infection with Gs/HK/437-4/99 (H5N1) than chickens and less virus is needed to kill quail than to kill chickens.

This study was supported by Public Health Research grants AI29680 and AI95357 from the National Institute of Allergy and Infectious Diseases, Wellcome Trust grant 057476/Z/99/Z, Cancer Center Support (CORE) grant CA-21765, and the American Lebanese Syrian Associated Charities. Support for Robert G. Webster to work in Hong Kong on this project was provided by the University of Hong Kong under the distinguished visiting scholars scheme.

We gratefully acknowledge the continued support of the officers of the Food and Environmental Hygiene Department, Hong Kong. We thank L. J. Zhang, P. Ghose, Jennifer Humberd, Patrick Seiler, C. Y. Cheung, and C. H. Lee for excellent technical support of this study, Alice Herren and Laurie Twit for preparation of the manuscript, and Julia Cay Jones for editorial assistance.