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Adaptation and transmission of a duck-origin avian influenza virus in poultry species
Virus Research 147 (2010) 40–46
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Adaptation and transmission of a duck-origin avian influenza virus in poultry species Jinling Li a,b , Heinrich zu Dohna b , Nichole L. Anchell a , Sean C. Adams a , Nguyet T. Dao a , Zheng Xing c , Carol J. Cardona a,c,∗ a b c
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA Center for Animal Disease Modeling and Surveillance, School of Veterinary Medicine, University of California, Davis, CA 95616, USA Veterinary Medicine Extension, University of California, Davis, CA 95616, USA
a r t i c l e
i n f o
Article history: Received 27 June 2009 Received in revised form 5 October 2009 Accepted 7 October 2009 Available online 14 October 2009 Keywords: Influenza A virus Low pathogenicity H11N9 Transmission Adaptation Duck Chicken
a b s t r a c t A duck-origin avian influenza virus (AIV) was used to study viral adaptation and transmission patterns in chickens (Gallus gallus domesticus) and Pekin ducks (Anas platyrhynchos domesticus). Inoculated birds were housed with naïve birds of the same species and all birds were monitored for infection. The inoculating duck virus was transmitted effectively by contact in both species. Viruses recovered from infected birds showed mutations as early as 1 or 3 days after inoculation in chickens and ducks, respectively. Amino acid substitutions in hemagglutinin (HA) or deletions in neuraminidase (NA) stalk regions were identified in chicken isolates, but only substitutions in HA were identified in duck isolates. HA substitution-containing viruses replicated more efficiently than those with NA stalk deletions. NA deletion mutants were not recovered from contact chickens, suggesting inefficient transmission. Amino acid substitutions in HA proteins appeared in pairs in chickens, but were independent in ducks, indicating adaptation in chickens. In addition, our findings showed that a duck-origin virus can rapidly adapt to chickens, suggesting that the emergence of new epidemic AIV can be rapid. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Avian influenza viruses (AIVs) belong to the Orthomyxoviridae and are enveloped viruses with a single-stranded, negative-sense RNA genome. The genetic material is composed of eight RNA segments, which encode two glycoproteins, the hemagglutinin (HA) and neuraminidase (NA), and nine other proteins (Lamb, 1989; Webster et al., 1992). All AIV are classified as type A influenza viruses and subtyped according to the combination of HA and NA (Fouchier et al., 2005; Kawaoka et al., 1990; Lamb, 1989; Rohm et al., 1996). Wild aquatic and migratory birds, from the Anseriformes and Charadriiformes orders, are natural reservoirs for AIV. They commonly display no clinical signs when infected
Abbreviations: LPAIV, low pathogenicity avian influenza virus; HA, hemagglutinin; NA, neuraminidase; HI, hemagglutination inhibition; BLP, base-line prevalence. ∗ Corresponding author at: Room 1383, Surge I, One Shields Avenue, University of California, Davis, CA 95616, USA. Tel.: +1 530 754 5041; fax: +1 530 752 7563. E-mail addresses: [email protected] (J. Li), [email protected] (H.z. Dohna), [email protected] (N.L. Anchell), [email protected] (S.C. Adams), [email protected] (N.T. Dao), [email protected] (Z. Xing), [email protected] (C.J. Cardona). 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.10.002
with AIV (Stallknecht and Shane, 1988; Webster et al., 1992), and most AIV are of low virulence in non-reservoir hosts. Two HA subtypes, H5 and H7, can become highly pathogenic in poultry. To protect humans and livestock from emerging viruses, it is crucial to understand the mechanisms by which viruses colonize and evolve in new host species. Viruses of different subtypes can infect poultry species with different efficiencies. Some subtypes, such as H5 (Nguyen et al., 2005; Perkins and Swayne, 2001), H6 (Kinde et al., 2003; Webby et al., 2003), H7 (Cecchinato et al., 2008), H9 (Choi et al., 2004) and H10 (Wood et al., 1996) have caused outbreaks in poultry. Other subtypes, such as H11 to H16, have only rarely been reported in poultry or hosts other than wild ducks and shorebirds (McCracken, 2006; VanDalen et al., 2008). To understand how AIV adapt from ducks to poultry species, we selected a viral subtype, H11N9, for which there are no published sequences that have the hallmarks of adaptation to chickens (Li et al., 2008; McCracken, 2006). However, seroconversion has been documented in waterfowl hunters and wildlife professionals (Gill et al., 2006). In this study, we investigated the adaptation and transmission patterns of a duck-origin AIV in chickens and Pekin ducks, and compared biological properties of the virus in the two hosts. The findings from this study provide insight in
J. Li et al. / Virus Research 147 (2010) 40–46
understanding the transmission patterns of wild bird AIV to poultry. 2. Materials and methods 2.1. Virus and experimental birds The virus used in this study (A/duck/WA/663/1997, H11N9) (hereafter, duck-origin or H11N9) was isolated from a captive mandarin duck (Aix galericulata). Experimental animals were Pekin ducks (Anas platyrhynchos domesticus) and domestic chickens (Gallus gallus domesticus). The Pekin is a domesticated breed derived from mallard ducks (Anas platyrhynchos) and has been frequently used in AIV research to understand viral adaptation, pathogenicity, transmission and for the development of vaccines (Londt et al., 2008; Pantin-Jackwood and Swayne, 2007; Steensels et al., 2009; Yee et al., 2009). Chickens are commercially important and globally the most prevalent poultry species. Studies have demonstrated that HPAIV H5N1 infection in humans and other mammalian species are strongly linked with spread among chicken flocks (Keawcharoen et al., 2004; Subbarao et al., 1998). Together, these two species are among the most important in avian influenza transmission cycles worldwide and, thus, were selected for these studies. 2.2. Animal studies Three animal experiments were conducted, one in ducks and two in chickens, to understand virus transmission from infected birds to naïve birds of the same species. Unless stated otherwise, experimental procedures were identical. The experimental animals included 25 commercially raised, 67-week-old, egg-producing white leghorn (W36 Hyline) hens and 20 three-week-old Pekin ducks, including 12 drakes and eight hens. All birds were tested for H11N9 sero-negativity before they were placed in isolation units with positive-pressure and filtered air supplies. The animals were housed in the controlled environment with an ambient temperature of 20–22 ◦ C. Fresh water and feed (Layena, Purina Mills, MO) were supplied ad libitum. The birds were given a day to acclimate to the new environment after placement. Five chickens and 10 ducks (six drakes and four hens) were designated as negative control birds and received 200 L of sterile PBS each via the intranasal route. Each experimental group consisted of 10 birds of a single species. Experimental groups were housed separately, one per isolation unit. Each bird was swabbed oropharyngeally and cloacally and the samples were tested prior to inoculation to assure that they were not infected with AIV. A 20% base-line prevalence (BLP) of AIV was established in the duck group, by inoculation of 2 of the 10 animals (six drakes and four hens) with 200 L of 107.1 TCID50 /mL (median tissue culture infectious dose) of H11N9 virus intranasally (i.n.). Twenty percent and 50% BLPs were established in the chicken groups; two or five chickens were inoculated with 200 L of 107.1 TCID50 /mL of virus per bird i.n. After inoculation, birds were immediately replaced into the experimental group. From the second day post inoculation (P.I.) (designated as day 1), all birds were swabbed oropharyngeally and cloacally daily over the course of the experiment (20 days for chickens and 21 days for ducks). Blood samples were collected on days 3, 6, 10, 13, 17 and 20 P.I. from chickens and days 5, 7, 11, 14, 18 and 21 P.I. from ducks. At the conclusion of each trial, all birds were humanely euthanized with an overdose of carbon dioxide. 2.3. Virological analysis Oropharyngeal and cloacal samples were tested for the presence of viruses to assess virus shedding patterns. The swab samples were inoculated into specific-pathogen-free (SPF) chicken embryos
41
following standard methods (WHO, 2002). Allantoic fluid samples were screened for AIV by testing hemagglutinating (HA) activity using 0.5% chicken blood (Colorado Serum Company, Denver, CO) as previously described (Li et al., 2008; WHO, 2002). In addition to virus isolation in eggs, swab samples from chickens and Pekin ducks were also inoculated onto Madin-Darby Canine Kidney cells (MDCK, ATCC, Manassas, VA) a day after the cells were seeded to determine viral titers by either plaque (Gaush and Smith, 1968) or TCID50 assay. Inoculated MDCK cells were incubated at 37 ± 0.5 ◦ C with 5% CO2 in serum-free Dulbecco’s Modified Eagle Medium (DMEM) supplemented with a 1× antibiotic/antimycotic solution (Invitrogen, Carlsbad, CA). Virus titers were calculated by counting plaques or the number of wells with cytopathic effect (CPE) and calculating plaque forming units (pfu/mL) (WHO, 2002) or HA activity (Reed and Muench, 1938). The viruses were grouped as HA mutants, NA deletion mutants or original inoculating virus based on their genotypes. In vivo growth kinetics were determined for each virus category using the titers obtained for each time point. The average titers over 3 days are presented in Fig. 2. The samples that were negative by egg inoculation, but positive for CPE or HA activity in MDCK cell cultures were further tested for the presence of two fragments of H11 gene using primers previously described (Li et al., 2008). The samples that were positive for either fragment or both were considered positive for H11N9 infection. These data were used for the calculation of transmission rate. Viruses collected from plaques were expanded in chicken embryos for genotyping. 2.4. Genetic analysis Viruses recovered from infected birds by isolation were tested for the H11 HA and N9 NA genes by RT-PCR and the amplicons used for partial genotyping. Virus preparations included direct inoculation of chicken embryos or MDCK cells, or additional expansion of virus in plaques. RNA extraction and RT-PCR using H11 and N9 gene-specific primers have been described (Hoffmann et al., 2001; Li et al., 2008). An additional primer pair for the N9 stalk region sequence, N9-1F (5 -ATGAATCCAAATCAGAAGATTCTATGAC-3 ) and N9-419R (5 GTTGTCCCTTGGCTGAGCATAGAACCTGCA-3 ) were designed for this study. The NA stalk PCR assay could detect N9 if the virus had a titer of >100.5 TCID50 . RT-PCR products were purified using Microcon filters following the manufacturer’s instructions (Millipore Corporation, Bedford, MA) and were sequenced using the amplification primers (Davis Sequencing Inc., Davis, CA). Nucleotide sequences were assembled using ContigExpress and the derived amino acids were aligned with AlignX of Vector NTI Advanced X (Invitrogen, Carlsbad, CA) for mutation assessment. 2.5. Statistical analysis of amino acid substitutions The number and distribution of amino acid substitutions in the HA proteins of the recovered H11N9 viruses were analyzed. The number of daily amino acid substitutions was determined by the number of amino acid differences between the sequences coming from a virus from the same bird on two consecutive days. The virus sequences that were first obtained from an individual bird and thus did not have a prior sequence for comparison were compared, to their putative source. Hence, the sequence of the first isolate from the inoculated birds was compared to the sequence of the inoculating virus and the sequences of the first isolates from the contact birds were compared to the virus with the most similar sequence in previously infected birds. Analysis is based on the assumption that if substitutions appear independently at a constant rate, the number of daily substitutions per bird would follow a Poisson distribution (Chiang, 1980) and that an association between different substitutions should lead to an overdispersed distribution such as
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J. Li et al. / Virus Research 147 (2010) 40–46
Fig. 1. Cumulative number of confirmed infected birds over time for the 20% baseline prevalence (BLP) chicken group (solid line), 50% BLP chicken group (dashed line) and the 20% BLP duck group (dotted line). Infection was determined by virus shedding for chickens and virus shedding and seroconversion for ducks.
a negative binomial distribution (Chiang, 1980; Rice, 1995). Therefore, a likelihood ratio test was used to determine, for each host, whether a negative binomial or a Poisson distribution was a better fit to the frequency distribution of daily substitution events. 2.6. Serological analysis Blood was taken from each bird 1 day prior to virus challenge and on designated days after inoculation as described in Section 2.2. Sera were separated from whole blood and tested for antigen-
specific antibody titers against the H11N9 inoculating virus or the viruses recovered from the infected chickens using a standard hemagglutination inhibition (HI) assay (Palmer et al., 1975; WHO, 2002). Since the HI assay may not reflect the true antibody levels of ducks due to the heterogeneity of IgY antibodies (Yoden et al., 1982), a homologous neutralization test was performed to determine the antibody titers using a plaque reduction assay (Gaush and Smith, 1968; Jahiel and Kilbourne, 1966; Zakay-Rones et al., 1980). Sera were diluted 1:5 and the inoculating virus (titer of 107 pfu/mL) was diluted 1:5000 in sterile phosphate buffered saline (PBS). Equal volumes of the diluted virus and serum were mixed to achieve final dilutions of 1:10,000 and 1:10 for virus and serum, respectively. The mixture was then incubated at 37 ◦ C for 1 h to allow antigen–antibody interactions to occur. Mixtures were serially diluted to 100 , 10−1 and 10−2 and then used to infect 1-day-old MDCK cells with duplicates. Viruses were allowed to interact with cells for 1 h with periodic rocking of the culture plates. The rest of the assay was carried out following the plaque assay procedure (Gaush and Smith, 1968). Sera collected prior to the inoculations were used as negative controls. The percent plaque reduction was calculated as follows: reduction % = (pfu/mL after neutralization with naïve serum − pfu/mL with antiserum)/pfu/mL with naïve serum. 3. Results 3.1. Virus transmission among chickens and ducks In the experiment, 100% of the contact birds were infected, as determined by the detection of viral RNA, isolation of virus or seroconversion to H11N9 (findings are summarized in Fig. 1). The two inoculated chickens in the 20% BLP group began shedding virus cloacally or oropharyngeally 1 or 2 days P.I. One bird (C-5; bird des-
Table 1 Nucleotide/amino acid changes in HA and deletion mutation in NA from chicken isolates. Infection route
Inoculated
Prevalence (%)
Bird ID
20
C-5 C-7
50
C-11
C-12 C-14 Contact
20
C-3 C-8
C-10 Input virus a b c d e
Nucleotide positions. Amino acid positions. Not sequenced. Full length form of NA stalk. Deleted form of NA stalk.
Days P.I.
2 2 3 4 5 6 7 8 1 2 3 4 6 1 2 1 3 3 5 6 7 9 10 3
NA deletion
Nucleotide position/amino acid position 332a /113b
463/144
625/198
721/230
853/274
– – Fd F F F F F De – D D D D D F F
t/Y t/Y t/Y c/Y c/Y t/Y t/Y t/Y c/Y c/Y c/Y – c/Y c/Y c/Y c/Y c/Y
g/G g/G g/G g/G g/G g/G g/G a/D g/G g/G g/G g/G g/G g/G g/G g/G g/G
c/A t/V t/V t/V t/V t/V t/V t/V c/A c/A c/A c/A c/A c/A c/A t/V t/V
g/S g/S g/S g/S g/S g/S g/S a/N g/S g/S g/S g/S g/S g/S g/S g/S g/S
c/S t/F t/F t/F t/F t/F t/F t/F c/S c/S c/S c/S c/S c/S c/S t/F t/F
F F F – F – F
t/Y c/Y c/Y c/Y c/Y c/Y c/Y
g/G g/G g/G g/G g/G g/G g/G
t/V t/V t/V t/V t/V t/V t/V
g/S g/S g/S g/S g/S g/S g/S
t/F t/F t/F t/F t/F t/F t/F
F
t/Y
g/G
c/A
g/S
c/S
c
J. Li et al. / Virus Research 147 (2010) 40–46
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Table 2 Nucleotide/amino acid changes in HA from Pekin duck isolates. Infection route
Inoculated
Bird ID
D-7
D-10
Contact
D-9
Days P.I.
361a /110b
721/230
771/247
800
962
1030/333
1038/336
3 4 5 3 4 5
c/T t/I c/T c/T c/T c/T
g/S g/S t/I g/S g/S g/S
a/T a/T g/A a/T a/T a/T
a/R a/R a/R a/R a/R t/R
c/Y c/Y t/Y c/Y c/Y c/Y
t/L t/L t/L c/P c/P c/P
g/V g/V g/V g/V g/V g/V
4 5 6 7
c/T c/T c/T c/T
t/I t/I t/I t/I
a/T a/T a/T a/T
a/R a/R a/R a/R
c/Y c/Y c/Y c/Y
t/L t/L t/L t/L
g/V a/I a/I a/I
c/T
g/S
a/T
a/R
c/Y
t/L
g/V
Input virus a b
Nucleotide position/amino acid position
Nucleotide positions. Amino acid positions.
ignations hereafter will be C for chicken and D for duck followed by a number for the individual bird) shed virus for 1 day while the other shed intermittently up to 13 days P.I. (C-7). Virus shedding among the contact chickens started on various days, from as early as day 1 (C-6) to as late as day 11 (C-2) after the inoculation of the index birds. Intermittent shedding was observed in all contact birds. In the chicken 50% BLP group, all naïve birds were infected by contact and the pattern of virus shedding was similar to that in the 20% BLP group. In the duck experiment, seven of eight naïve ducks became infected through contact (one duck died on the second day, unrelated to infection; Fig. 1). Both inoculated ducks (D-7 and D-10) started shedding the virus oropharyngeally on day 3 P.I. One duck (D-9), infected through contact, began shedding on day 4 P.I. No virus was detected from oropharyngeal or cloacal swabs collected from the negative control chickens or ducks. 3.2. Genetic and phenotypic adaptation Twenty-four isolates were recovered from eight infected chickens at various time points during the experiment. Five of the chickens were infected by inoculation and three by contact. Virus was not recovered from all of the infected chickens, presumably
Fig. 2. Fitted (lines) and observed (bars) frequency distribution of daily amino acid substitutions for chickens (triangles and shaded-striped bars) and ducks (circles and striped bars). The best fitting distribution is Poisson for ducks and negative binomial for chickens.
due to the low titers in those birds and intermittent shedding. Nucleotide sequences of HA and NA stalk regions of all 24 isolates were of the H11N9 subtype. A comparison of the HA genes between the recovered viruses and the inoculating virus identified five changes within the 1449 bases, one synonymous and four non-synonymous (numbering for both nucleotide and amino acid sequences are based on that of the H11 HA consensus; Table 1). The inoculating virus has Ala and Ser at positions 198 and 274 of HA protein, respectively, whereas 17 of the 24 recovered chicken isolates had Val198 and Phe274 instead (Table 1). Viruses from all contact chickens and two of the five inoculated chickens had Val198 and Phe274 (Table 1). One of the inoculated chickens (C-7) had two additional substitutions of Gly144Asp and Ser230Asn in the HA protein on day 8 P.I. Ten viruses were isolated from three infected Pekin ducks and mutations in the HA genes were detected in seven positions, five of which were non-synonymous (Table 2). One of the six substitutions (amino acid position 230) was common to the viruses recovered from one inoculated (D-7) and one contact (D-9) duck. Substitutions at amino acid positions 110, 247, 333 and 336 were unique for each individual isolates (Table 2). The number of daily nucleotide substitutions in the HA gene deviated significantly from a Poisson distribution for chickens (P = 0.02, likelihood ratio test) but not for ducks (Fig. 2). Viruses from two inoculated chickens (C-11 and C-12) had a 90-nucleotide deletion in the stalk region of the NA gene, representing 30 amino acid residues (positions 44–73, numbering of
Fig. 3. In vivo replication of the H11N9 viruses in inoculated chickens. , inoculating virus from C-5; , HA mutants from C-7 and C-14; , NA stalk deletion mutants from C-11 and C-12. Each point represents a 3-day rolling average titer.
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J. Li et al. / Virus Research 147 (2010) 40–46
Table 3 Serum conversion in chickens and Pekin ducks (HI unit). Chicken
Days post inoculation
C-5a C-7a C-11a C-12a C-14a Duck
D-3 D-7a D-8 D-9 D-10a
0
3
6
10
13
17
0 0 0 0 0
0 0 0 0 0
0 4 4 8 8
NT 8 32 2 2
4 32 16 0 0
4 0 16 0 0
Days post inoculation 0
5
7
11
14
18
0 0 0 0 0
0 0 0 0 0
0 0 0 0 16
0 0 4 16 32
0 0 2 4 16
4 4 NT 4 16
Fig. 4. Antigen-specific antibody against the inoculating H11N9 virus in Pekin ducks measured by plaque reduction assay. Numbers represent individual ducks. Asterisks indicate inoculated birds.
NT, not tested. a Inoculated birds.
A/duck/WA/663/97 NA protein) from the first day P.I. (Table 1). No NA stalk deletion was detected in the inoculating virus when tested by RT-PCR (data not shown). None of the seven viruses recovered from three chickens infected by contact had deletions in the NA stalk. No viruses were isolated from contact birds in the 50% BLP group from which NA stalk deleted viruses persisted for as long as 6 days. Viruses recovered from two infected and one contact duck had intact NA stalk regions. Viruses rescued from infected chickens could be divided into two groups based on their patterns of mutation. One group of viruses had amino acid substitutions in HA proteins (Table 1, C3, C-7, C-8, C-10 and C-14). Another group of viruses contained NA stalk deletions (Table 1, C-11 and C-12). The HA substitution viruses replicated to higher levels and for longer periods of time than the NA stalk deletion mutants in vivo, and both types of mutants replicated more efficiently than the inoculating virus (Fig. 3). 3.3. Host responses to virus infection The two inoculated chickens in the 20% BLP group (C-5 and C-7) and three in the 50% BLP group (C-11, C-12 and C-14) produced low levels of antibodies, ranging from 4 to 32 HI units against the inoculating virus whereas antibodies were not detected in any contact chickens (Table 3). Sera from the contact chickens did not neutralize the inoculating virus. One inoculated duck (D-10) displayed an antibody response on day 7 P.I. and produced the highest antibody titer of 32 HI units. In contrast to D-10, another inoculated duck, D7, had an antibody level of 4 HI units on day 18 P.I. A contact duck (D-9) from which we were able to isolate viruses, had antibody titer comparable to that of D-10. D-3 and D-8 had detectable antibodies in response to infection (Table 3), although no virus was recovered from them. In addition to the HI results, the plaque reduction assay (PRA) showed that the viruses elicited detectable humoral responses in Pekin ducks (Fig. 4). Sera from D-7, D-9 and D-10 neutralized more than 90% of the inoculating virus, which confirmed the HI results (Table 3). Sera from D-1, D-5, D-6 and D-8 neutralized more than 25%, but less than 80% of the inoculating virus. The detection of seroconversion by PRA and HI were not equivalent in these experiments. Sera from D-1, D-5 and D-6 were positive by PRA and negative for HI, while duck D-3 was negative by PRA and positive by HI (Table 3). Changes in HA protein during the course of infection may influence the antibody profile. A cross reactivity analysis was performed using virus and serum samples collected from two chickens at different time points. The viruses were from C-7 (inoculated chicken)
Fig. 5. Cross reactivity between viruses recovered from infected chickens and sera collected at various time points. (Legend denotes viruses isolated from chickens on specified days.)
on days 3, 4 and 8 P.I. and from C-8 (contact chicken) on day 5 P.I. (Fig. 5). Sera were collected from C-7 on days 6, 10 and 13 P.I. and C-8 on day 13 P.I. The antibodies from C-7 on day 6 P.I. only neutralized the inoculating virus but not viruses recovered at later days. Sera from C-7 at later time points neutralized all the viruses (Fig. 5). Serum collected from C-8 (contact chicken) on day 13 P.I. neutralized all tested viruses isolated 3 days after initial inoculation, but had no neutralizing activity against the inoculating virus. 4. Discussion There are a few AIV subtypes with no reported outbreaks in poultry, suggesting that subtype specific constraints may either prevent their infection of, or transmission between, domestic chickens and ducks. To explore this possibility, we used an H11N9 LPAIV of duck origin to infect two poultry species, chicken (Gallus gallus domesticus) and Pekin duck (Anas platyrhynchos domesticus). These studies showed that the original duck virus was transmissible among both hosts by direct contact. In both hosts, 100% of the inoculated and contact birds were infected during the study, as determined by either virus detection or seroconversion. Our findings demonstrated that genetic changes in the virus genes occurred at different positions and at different rates in chickens and in ducks. The pattern of nucleotide substitutions in HA genes of the duck isolates are consistent with a model that assumes substitutions appear independently of each other and at a constant rate (Rice, 1995). In contrast, nucleotide substitutions in HA genes among chicken isolates appeared to be non-random, suggesting
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specific selection pressures in chickens, presumably related to host adaptation. The genetic changes associated with the adaptation of an AIV to domestic avian hosts have been reported for other subtypes (Hossain et al., 2008), but not previously for H11N9. In these studies, we found that two amino acid substitutions in the HA protein (A198V and S274F) were consistent among viruses isolated from chickens. Viruses from two of the inoculated and all contact chickens had the same substitutions. Alanine and serine at these two positions from the inoculating virus were found common among all 64 currently published H11 HA proteins (McCracken, 2006). This suggests that chickens impose a selection regime on the H11 HA gene that is different from the selection processes in reservoir avian hosts. Amino acid changes in the HA proteins were more random and less persistent in the isolates from ducks compared to those from chickens (Tables 1 and 2). Additionally, the HA genes of the H11N9 virus in ducks had a continuous low rate of nucleotide substitutions, whereas, in chickens, changes occurred early and then remained throughout the experimental period. A possible explanation for this pattern is that the observed nucleotide substitutions in the H11 HA gene in ducks were results of random fluctuation in the frequency of different variants. In contrast, the selection by chickens for a particular virus variant reduces this fluctuation, and hence the observed lower daily substitution rate. We observed that the same synonymous substitution appeared together with two amino acid substitutions in the HA proteins of the viruses isolated from three inoculated chickens housed separately. Therefore, it is reasonable to assume that pre-existing quasi-species in the inoculum played an important role in the rapid adaptation of H11N9 to chickens. Alternatively, it is possible that rapid and frequent mutations could occur during the virus amplification processes in this host species. The stalk deletions in the NA proteins in viruses recovered from two inoculated chickens are not found in any of the 88 N9 NA sequences currently available in public databases (McCracken, 2006). There is no evidence to support the pre-existence of the stalk deletion in the inoculating virus after testing multiple batches of virus preparations with various dilutions using RT-PCR (data not shown), although NA stalk deleted virus below the detection limit of the test could have been present in the inoculum. We conclude that NA stalk deletion, which has been linked previously to the virus isolates from gallinaceous birds (Giannecchini et al., 2006), either developed rapidly in this AIV through new mutations or was rapidly selected. The appearance of an NA stalk deletion was not a frequent or consistent event in this study. Among 17 chickens inoculated with A/duck/WA/663/97 included in this report and a previous study (Li et al., 2008), only two birds (12%) shed viruses with NA stalk deletions. A comparison of in vivo replication kinetics between the NA deletion and HA substitution mutants in these studies demonstrated that the HA mutants replicated better and were transmitted by contact to other chickens. In contrast, the NA stalk deletion mutants replicated better in chickens than the inoculating virus, but less efficiently than the HA mutant viruses. Although it is likely that NA stalk deletion resulted in a virus more fit for growth in chickens, it clearly was not as critical as the HA substitutions. None of the amino acid substitutions in the H11 HA protein changed the pattern of glycosylation or receptor-binding sites. However, serological analysis suggested that the changes may have affected antibody neutralization (Fig. 5). One possible explanation is that virus adapting to new hosts undergoes continuous changes to evade antibodies, resulting in viruses that are less effectively neutralized during an infection. This is supported by the fact that viruses continued to replicate in the chickens after antibodies were detected, indicating an incomplete immune clearance. In addition to escaping humoral immune responses, mutations in the HA and
45
NA proteins may also affect the efficiency of cell-mediated immune responses, which play a major role in AI pathogenesis (Swayne and Kapczynski, 2008). This study explored viral adaptation to poultry species by using a duck-origin isolate of a subtype rarely detected in domestic avian species. The data provided specific information on genetic changes and phenotypes of intermediate viruses isolated during adaptation. Based on these studies, it appears that there are few or no barriers to viral adaptation, even in subtypes that have not caused outbreaks in poultry. This provides critical testimony for the need for measures and practices that reduce contact between poultry and free-flying birds to prevent disease and to slow viral evolution. Acknowledgements Funding for these studies was provided by the Center for Food and Animal Health at the University of California, Davis. We would like to express our heartfelt appreciation to Dr. Zengqi Yang, Phuong Dao, Jerome Anunciacion, Heather Krabbenhoft, MaiLee Yang, and Kou Yang for their expert technical support. References Cecchinato, M., Bonfanti, L., Marangon, S., Terregino, C., Monne, I., Luppi, A., 2008. Low pathogenic avian influenza in Italy. Vet. Rec. 162 (2), 64. Chiang, C.L., 1980. An Introduction to Stochastic Processes and their Applications. Krieger Publishing Company, New York, USA. Choi, Y.K., Ozaki, H., Webby, R.J., Webster, R.G., Peiris, J.S., Poon, L., Butt, C., Leung, Y.H., Guan, Y., 2004. Continuing evolution of H9N2 influenza viruses in Southeastern China. J. Virol. 78 (16), 8609–8614. Fouchier, R.A., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B., Osterhaus, A.D., 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J. Virol. 79 (5), 2814–2822. Gaush, C.R., Smith, T.F., 1968. Replication and plaque assay of influenza virus in an established line of canine kidney cells. Appl. Microbiol. 16 (4), 588–594. Giannecchini, S., Campitelli, L., Calzoletti, L., De Marco, M.A., Azzi, A., Donatelli, I., 2006. Comparison of in vitro replication features of H7N3 influenza viruses from wild ducks and turkeys: potential implications for interspecies transmission. J. Gen. Virol. 87 (Pt 1), 171–175. Gill, J.S., Webby, R., Gilchrist, M.J., Gray, G.C., 2006. Avian influenza among waterfowl hunters and wildlife professionals. Emerg. Infect. Dis. 12 (8), 1284–1286. Hoffmann, E., Stech, J., Guan, Y., Webster, R.G., Perez, D.R., 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146 (12), 2275–2289. Hossain, M.J., Hickman, D., Perez, D.R., 2008. Evidence of expanded host range and mammalian-associated genetic changes in a duck H9N2 influenza virus following adaptation in quail and chickens. PLoS ONE 3 (9), e3170. Jahiel, R.I., Kilbourne, E.D., 1966. Reduction in plaque size and reduction in plaque number as differing indices of influenza virus-antibody reactions. J. Bacteriol. 92 (5), 1521–1534. Kawaoka, Y., Yamnikova, S., Chambers, T.M., Lvov, D.K., Webster, R.G., 1990. Molecular characterization of a new hemagglutinin, subtype H14, of influenza A virus. Virology 179 (2), 759–767. Keawcharoen, J., Oraveerakul, K., Kuiken, T., Fouchier, R.A., Amonsin, A., Payungporn, S., Noppornpanth, S., Wattanodorn, S., Theambooniers, A., Tantilertcharoen, R., Pattanarangsan, R., Arya, N., Ratanakorn, P., Osterhaus, D.M., Poovorawan, Y., 2004. Avian influenza H5N1 in tigers and leopards. Emerg. Infect. Dis. 10 (12), 2189–2191. Kinde, H., Read, D.H., Daft, B.M., Hammarlund, M., Moore, J., Uzal, F., Mukai, J., Woolcock, P., 2003. The occurrence of avian influenza A subtype H6N2 in commercial layer flocks in Southern California (2000-02): clinicopathologic findings. Avian Dis. 47 (3 Suppl.), 1214–1218. Lamb, R.A., 1989. Genes and proteins of the influenza viruses. In: Krug, R.M., Fraenkel-Conrat, H., Wagner, R.R. (Eds.), The Influenza Viruses. Plenum Press, New York, pp. 1–88. Li, J., Cardona, C.J., Xing, Z., Woolcock, P.R., 2008. Genetic and phenotypic characterization of a low-pathogenicity avian influenza H11N9 virus. Arch. Virol. 153 (10), 1899–1908. Londt, B.Z., Nunez, A., Banks, J., Nili, H., Johnson, L.K., Alexander, D.J., 2008. Pathogenesis of highly pathogenic avian influenza A/turkey/Turkey/1/2005 H5N1 in Pekin ducks (Anas platyrhynchos) infected experimentally. Avian Pathol. 37 (6), 619–627. McCracken, C., 2006. The Influenza Sequence Database. Los Alamos National Laboratory. Nguyen, D.C., Uyeki, T.M., Jadhao, S., Maines, T., Shaw, M., Matsuoka, Y., Smith, C., Rowe, T., Lu, X., Hall, H., Xu, X., Balish, A., Klimov, A., Tumpey, T.M., Swayne, D.E., Huynh, L.P., Nghiem, H.K., Nguyen, H.H., Hoang, L.T., Cox, N.J., Katz, J.M., 2005. Isolation and characterization of avian influenza viruses, including highly
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pathogenic H5N1, from poultry in live bird markets in Hanoi, Vietnam, in 2001. J. Virol. 79 (7), 4201–4212. Palmer, D., Coleman, M., Dowdle, W., Schild, G., 1975. Advanced Laboratory Techniques for Influenza Diagnosis. U.S. Department of Health, Education, and Welfare (Immunology Series No. 6). Pantin-Jackwood, M.J., Swayne, D.E., 2007. Pathobiology of Asian highly pathogenic avian influenza H5N1 virus infections in ducks. Avian Dis. 51 (1 Suppl.), 250–259. Perkins, L.E., Swayne, D.E., 2001. Pathobiology of A/chicken/Hong Kong/220/97 (H5N1) avian influenza virus in seven gallinaceous species. Vet. Pathol. 38 (2), 149–164. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493–497. Rice, J.A., 1995. Mathematical Statistics & Data Analysis, 2nd ed. Duxbury Press, Belmont, CA, USA. Rohm, C., Zhou, N., Suss, J., Mackenzie, J., Webster, R.G., 1996. Characterization of a novel influenza hemagglutinin, H15: criteria for determination of influenza A subtypes. Virology 217 (2), 508–516. Stallknecht, D.E., Shane, S.M., 1988. Host range of avian influenza virus in free-living birds. Vet. Res. Commun. 12 (2–3), 125–141. Steensels, M., Bublot, M., Van Borm, S., De Vriese, J., Lambrecht, B., Richard-Mazet, A., Chanavat-Bizzini, S., Duboeuf, M., Le Gros, F.X., van den Berg, T., 2009. Primeboost vaccination with a fowlpox vector and an inactivated avian influenza vaccine is highly immunogenic in Pekin ducks challenged with Asian H5N1 HPAI. Vaccine 27 (5), 646–654. Subbarao, K., Klimov, A., Katz, J., Regnery, H., Lim, W., Hall, H., Perdue, M., Swayne, D., Bender, C., Huang, J., Hemphill, M., Rowe, T., Shaw, M., Xu, X., Fukuda, K., Cox, N., 1998. Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279 (5349), 393–396.
Swayne, D.E., Kapczynski, D., 2008. Strategies and challenges for eliciting immunity against avian influenza virus in birds. Immunol. Rev. 225, 314– 331. VanDalen, K.K., Anderson, T.D., Killian, M.L., Pedersen, J.C., Franklin, A.B., Piaggio, A.J., 2008. Increased detection of influenza A H16 in the United States. Arch. Virol. 153 (10), 1981–1983. Webby, R.J., Woolcock, P.R., Krauss, S.L., Walker, D.B., Chin, P.S., Shortridge, K.F., Webster, R.G., 2003. Multiple genotypes of nonpathogenic H6N2 influenza viruses isolated from chickens in California. Avian Dis. 47 (3 Suppl.), 905– 910. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56 (1), 152–179. WHO, 2002. In: C.D.S.a. Response (Ed.), WHO Manual on Animal Influenza Diagnosis and Surveillance, 5th ed. WHO/CDC/CSR/NCS. Wood, G.W., Banks, J., Strong, I., Parsons, G., Alexander, D.J., 1996. An avian influenza virus of H10 subtype that is highly pathogenic for chickens, but lacks multiple basic amino acids at the haemagglutinin cleavage site. Avian Pathol. 25 (4), 799–806. Yee, K.S., Cardona, C.J., Carpenter, T.E., 2009. Transmission of low-pathogenicity avian influenza virus of subtype H6N2 from chickens to Pekin ducks and Japanese quail (Coturnix coturnix japonica). Avian Pathol. 38 (1), 59–64. Yoden, S., Kida, H., Yanagawa, R., 1982. An avian influenza virus of which infectivity is neutralized by antisera lacking hemagglutination-inhibition activity. Brief report. Arch. Virol. 74 (2–3), 205–210. Zakay-Rones, Z., Margalith, E., Levy, R., Katz, E., 1980. A sensitive plaque inhibition technique for assay of antibodies to influenza virus: use to detect previous antigenic priming with influenza viruses. J. Virol. Methods 1 (6), 355–360.
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Adaptation and transmission of a duck-origin avian influenza virus in poultry species Jinling Li a,b , Heinrich zu Dohna b , Nichole L. Anchell a , Sean C. Adams a , Nguyet T. Dao a , Zheng Xing c , Carol J. Cardona a,c,∗ a b c
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA Center for Animal Disease Modeling and Surveillance, School of Veterinary Medicine, University of California, Davis, CA 95616, USA Veterinary Medicine Extension, University of California, Davis, CA 95616, USA
a r t i c l e
i n f o
Article history: Received 27 June 2009 Received in revised form 5 October 2009 Accepted 7 October 2009 Available online 14 October 2009 Keywords: Influenza A virus Low pathogenicity H11N9 Transmission Adaptation Duck Chicken
a b s t r a c t A duck-origin avian influenza virus (AIV) was used to study viral adaptation and transmission patterns in chickens (Gallus gallus domesticus) and Pekin ducks (Anas platyrhynchos domesticus). Inoculated birds were housed with naïve birds of the same species and all birds were monitored for infection. The inoculating duck virus was transmitted effectively by contact in both species. Viruses recovered from infected birds showed mutations as early as 1 or 3 days after inoculation in chickens and ducks, respectively. Amino acid substitutions in hemagglutinin (HA) or deletions in neuraminidase (NA) stalk regions were identified in chicken isolates, but only substitutions in HA were identified in duck isolates. HA substitution-containing viruses replicated more efficiently than those with NA stalk deletions. NA deletion mutants were not recovered from contact chickens, suggesting inefficient transmission. Amino acid substitutions in HA proteins appeared in pairs in chickens, but were independent in ducks, indicating adaptation in chickens. In addition, our findings showed that a duck-origin virus can rapidly adapt to chickens, suggesting that the emergence of new epidemic AIV can be rapid. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Avian influenza viruses (AIVs) belong to the Orthomyxoviridae and are enveloped viruses with a single-stranded, negative-sense RNA genome. The genetic material is composed of eight RNA segments, which encode two glycoproteins, the hemagglutinin (HA) and neuraminidase (NA), and nine other proteins (Lamb, 1989; Webster et al., 1992). All AIV are classified as type A influenza viruses and subtyped according to the combination of HA and NA (Fouchier et al., 2005; Kawaoka et al., 1990; Lamb, 1989; Rohm et al., 1996). Wild aquatic and migratory birds, from the Anseriformes and Charadriiformes orders, are natural reservoirs for AIV. They commonly display no clinical signs when infected
Abbreviations: LPAIV, low pathogenicity avian influenza virus; HA, hemagglutinin; NA, neuraminidase; HI, hemagglutination inhibition; BLP, base-line prevalence. ∗ Corresponding author at: Room 1383, Surge I, One Shields Avenue, University of California, Davis, CA 95616, USA. Tel.: +1 530 754 5041; fax: +1 530 752 7563. E-mail addresses: [email protected] (J. Li), [email protected] (H.z. Dohna), [email protected] (N.L. Anchell), [email protected] (S.C. Adams), [email protected] (N.T. Dao), [email protected] (Z. Xing), [email protected] (C.J. Cardona). 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.10.002
with AIV (Stallknecht and Shane, 1988; Webster et al., 1992), and most AIV are of low virulence in non-reservoir hosts. Two HA subtypes, H5 and H7, can become highly pathogenic in poultry. To protect humans and livestock from emerging viruses, it is crucial to understand the mechanisms by which viruses colonize and evolve in new host species. Viruses of different subtypes can infect poultry species with different efficiencies. Some subtypes, such as H5 (Nguyen et al., 2005; Perkins and Swayne, 2001), H6 (Kinde et al., 2003; Webby et al., 2003), H7 (Cecchinato et al., 2008), H9 (Choi et al., 2004) and H10 (Wood et al., 1996) have caused outbreaks in poultry. Other subtypes, such as H11 to H16, have only rarely been reported in poultry or hosts other than wild ducks and shorebirds (McCracken, 2006; VanDalen et al., 2008). To understand how AIV adapt from ducks to poultry species, we selected a viral subtype, H11N9, for which there are no published sequences that have the hallmarks of adaptation to chickens (Li et al., 2008; McCracken, 2006). However, seroconversion has been documented in waterfowl hunters and wildlife professionals (Gill et al., 2006). In this study, we investigated the adaptation and transmission patterns of a duck-origin AIV in chickens and Pekin ducks, and compared biological properties of the virus in the two hosts. The findings from this study provide insight in
J. Li et al. / Virus Research 147 (2010) 40–46
understanding the transmission patterns of wild bird AIV to poultry. 2. Materials and methods 2.1. Virus and experimental birds The virus used in this study (A/duck/WA/663/1997, H11N9) (hereafter, duck-origin or H11N9) was isolated from a captive mandarin duck (Aix galericulata). Experimental animals were Pekin ducks (Anas platyrhynchos domesticus) and domestic chickens (Gallus gallus domesticus). The Pekin is a domesticated breed derived from mallard ducks (Anas platyrhynchos) and has been frequently used in AIV research to understand viral adaptation, pathogenicity, transmission and for the development of vaccines (Londt et al., 2008; Pantin-Jackwood and Swayne, 2007; Steensels et al., 2009; Yee et al., 2009). Chickens are commercially important and globally the most prevalent poultry species. Studies have demonstrated that HPAIV H5N1 infection in humans and other mammalian species are strongly linked with spread among chicken flocks (Keawcharoen et al., 2004; Subbarao et al., 1998). Together, these two species are among the most important in avian influenza transmission cycles worldwide and, thus, were selected for these studies. 2.2. Animal studies Three animal experiments were conducted, one in ducks and two in chickens, to understand virus transmission from infected birds to naïve birds of the same species. Unless stated otherwise, experimental procedures were identical. The experimental animals included 25 commercially raised, 67-week-old, egg-producing white leghorn (W36 Hyline) hens and 20 three-week-old Pekin ducks, including 12 drakes and eight hens. All birds were tested for H11N9 sero-negativity before they were placed in isolation units with positive-pressure and filtered air supplies. The animals were housed in the controlled environment with an ambient temperature of 20–22 ◦ C. Fresh water and feed (Layena, Purina Mills, MO) were supplied ad libitum. The birds were given a day to acclimate to the new environment after placement. Five chickens and 10 ducks (six drakes and four hens) were designated as negative control birds and received 200 L of sterile PBS each via the intranasal route. Each experimental group consisted of 10 birds of a single species. Experimental groups were housed separately, one per isolation unit. Each bird was swabbed oropharyngeally and cloacally and the samples were tested prior to inoculation to assure that they were not infected with AIV. A 20% base-line prevalence (BLP) of AIV was established in the duck group, by inoculation of 2 of the 10 animals (six drakes and four hens) with 200 L of 107.1 TCID50 /mL (median tissue culture infectious dose) of H11N9 virus intranasally (i.n.). Twenty percent and 50% BLPs were established in the chicken groups; two or five chickens were inoculated with 200 L of 107.1 TCID50 /mL of virus per bird i.n. After inoculation, birds were immediately replaced into the experimental group. From the second day post inoculation (P.I.) (designated as day 1), all birds were swabbed oropharyngeally and cloacally daily over the course of the experiment (20 days for chickens and 21 days for ducks). Blood samples were collected on days 3, 6, 10, 13, 17 and 20 P.I. from chickens and days 5, 7, 11, 14, 18 and 21 P.I. from ducks. At the conclusion of each trial, all birds were humanely euthanized with an overdose of carbon dioxide. 2.3. Virological analysis Oropharyngeal and cloacal samples were tested for the presence of viruses to assess virus shedding patterns. The swab samples were inoculated into specific-pathogen-free (SPF) chicken embryos
41
following standard methods (WHO, 2002). Allantoic fluid samples were screened for AIV by testing hemagglutinating (HA) activity using 0.5% chicken blood (Colorado Serum Company, Denver, CO) as previously described (Li et al., 2008; WHO, 2002). In addition to virus isolation in eggs, swab samples from chickens and Pekin ducks were also inoculated onto Madin-Darby Canine Kidney cells (MDCK, ATCC, Manassas, VA) a day after the cells were seeded to determine viral titers by either plaque (Gaush and Smith, 1968) or TCID50 assay. Inoculated MDCK cells were incubated at 37 ± 0.5 ◦ C with 5% CO2 in serum-free Dulbecco’s Modified Eagle Medium (DMEM) supplemented with a 1× antibiotic/antimycotic solution (Invitrogen, Carlsbad, CA). Virus titers were calculated by counting plaques or the number of wells with cytopathic effect (CPE) and calculating plaque forming units (pfu/mL) (WHO, 2002) or HA activity (Reed and Muench, 1938). The viruses were grouped as HA mutants, NA deletion mutants or original inoculating virus based on their genotypes. In vivo growth kinetics were determined for each virus category using the titers obtained for each time point. The average titers over 3 days are presented in Fig. 2. The samples that were negative by egg inoculation, but positive for CPE or HA activity in MDCK cell cultures were further tested for the presence of two fragments of H11 gene using primers previously described (Li et al., 2008). The samples that were positive for either fragment or both were considered positive for H11N9 infection. These data were used for the calculation of transmission rate. Viruses collected from plaques were expanded in chicken embryos for genotyping. 2.4. Genetic analysis Viruses recovered from infected birds by isolation were tested for the H11 HA and N9 NA genes by RT-PCR and the amplicons used for partial genotyping. Virus preparations included direct inoculation of chicken embryos or MDCK cells, or additional expansion of virus in plaques. RNA extraction and RT-PCR using H11 and N9 gene-specific primers have been described (Hoffmann et al., 2001; Li et al., 2008). An additional primer pair for the N9 stalk region sequence, N9-1F (5 -ATGAATCCAAATCAGAAGATTCTATGAC-3 ) and N9-419R (5 GTTGTCCCTTGGCTGAGCATAGAACCTGCA-3 ) were designed for this study. The NA stalk PCR assay could detect N9 if the virus had a titer of >100.5 TCID50 . RT-PCR products were purified using Microcon filters following the manufacturer’s instructions (Millipore Corporation, Bedford, MA) and were sequenced using the amplification primers (Davis Sequencing Inc., Davis, CA). Nucleotide sequences were assembled using ContigExpress and the derived amino acids were aligned with AlignX of Vector NTI Advanced X (Invitrogen, Carlsbad, CA) for mutation assessment. 2.5. Statistical analysis of amino acid substitutions The number and distribution of amino acid substitutions in the HA proteins of the recovered H11N9 viruses were analyzed. The number of daily amino acid substitutions was determined by the number of amino acid differences between the sequences coming from a virus from the same bird on two consecutive days. The virus sequences that were first obtained from an individual bird and thus did not have a prior sequence for comparison were compared, to their putative source. Hence, the sequence of the first isolate from the inoculated birds was compared to the sequence of the inoculating virus and the sequences of the first isolates from the contact birds were compared to the virus with the most similar sequence in previously infected birds. Analysis is based on the assumption that if substitutions appear independently at a constant rate, the number of daily substitutions per bird would follow a Poisson distribution (Chiang, 1980) and that an association between different substitutions should lead to an overdispersed distribution such as
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J. Li et al. / Virus Research 147 (2010) 40–46
Fig. 1. Cumulative number of confirmed infected birds over time for the 20% baseline prevalence (BLP) chicken group (solid line), 50% BLP chicken group (dashed line) and the 20% BLP duck group (dotted line). Infection was determined by virus shedding for chickens and virus shedding and seroconversion for ducks.
a negative binomial distribution (Chiang, 1980; Rice, 1995). Therefore, a likelihood ratio test was used to determine, for each host, whether a negative binomial or a Poisson distribution was a better fit to the frequency distribution of daily substitution events. 2.6. Serological analysis Blood was taken from each bird 1 day prior to virus challenge and on designated days after inoculation as described in Section 2.2. Sera were separated from whole blood and tested for antigen-
specific antibody titers against the H11N9 inoculating virus or the viruses recovered from the infected chickens using a standard hemagglutination inhibition (HI) assay (Palmer et al., 1975; WHO, 2002). Since the HI assay may not reflect the true antibody levels of ducks due to the heterogeneity of IgY antibodies (Yoden et al., 1982), a homologous neutralization test was performed to determine the antibody titers using a plaque reduction assay (Gaush and Smith, 1968; Jahiel and Kilbourne, 1966; Zakay-Rones et al., 1980). Sera were diluted 1:5 and the inoculating virus (titer of 107 pfu/mL) was diluted 1:5000 in sterile phosphate buffered saline (PBS). Equal volumes of the diluted virus and serum were mixed to achieve final dilutions of 1:10,000 and 1:10 for virus and serum, respectively. The mixture was then incubated at 37 ◦ C for 1 h to allow antigen–antibody interactions to occur. Mixtures were serially diluted to 100 , 10−1 and 10−2 and then used to infect 1-day-old MDCK cells with duplicates. Viruses were allowed to interact with cells for 1 h with periodic rocking of the culture plates. The rest of the assay was carried out following the plaque assay procedure (Gaush and Smith, 1968). Sera collected prior to the inoculations were used as negative controls. The percent plaque reduction was calculated as follows: reduction % = (pfu/mL after neutralization with naïve serum − pfu/mL with antiserum)/pfu/mL with naïve serum. 3. Results 3.1. Virus transmission among chickens and ducks In the experiment, 100% of the contact birds were infected, as determined by the detection of viral RNA, isolation of virus or seroconversion to H11N9 (findings are summarized in Fig. 1). The two inoculated chickens in the 20% BLP group began shedding virus cloacally or oropharyngeally 1 or 2 days P.I. One bird (C-5; bird des-
Table 1 Nucleotide/amino acid changes in HA and deletion mutation in NA from chicken isolates. Infection route
Inoculated
Prevalence (%)
Bird ID
20
C-5 C-7
50
C-11
C-12 C-14 Contact
20
C-3 C-8
C-10 Input virus a b c d e
Nucleotide positions. Amino acid positions. Not sequenced. Full length form of NA stalk. Deleted form of NA stalk.
Days P.I.
2 2 3 4 5 6 7 8 1 2 3 4 6 1 2 1 3 3 5 6 7 9 10 3
NA deletion
Nucleotide position/amino acid position 332a /113b
463/144
625/198
721/230
853/274
– – Fd F F F F F De – D D D D D F F
t/Y t/Y t/Y c/Y c/Y t/Y t/Y t/Y c/Y c/Y c/Y – c/Y c/Y c/Y c/Y c/Y
g/G g/G g/G g/G g/G g/G g/G a/D g/G g/G g/G g/G g/G g/G g/G g/G g/G
c/A t/V t/V t/V t/V t/V t/V t/V c/A c/A c/A c/A c/A c/A c/A t/V t/V
g/S g/S g/S g/S g/S g/S g/S a/N g/S g/S g/S g/S g/S g/S g/S g/S g/S
c/S t/F t/F t/F t/F t/F t/F t/F c/S c/S c/S c/S c/S c/S c/S t/F t/F
F F F – F – F
t/Y c/Y c/Y c/Y c/Y c/Y c/Y
g/G g/G g/G g/G g/G g/G g/G
t/V t/V t/V t/V t/V t/V t/V
g/S g/S g/S g/S g/S g/S g/S
t/F t/F t/F t/F t/F t/F t/F
F
t/Y
g/G
c/A
g/S
c/S
c
J. Li et al. / Virus Research 147 (2010) 40–46
43
Table 2 Nucleotide/amino acid changes in HA from Pekin duck isolates. Infection route
Inoculated
Bird ID
D-7
D-10
Contact
D-9
Days P.I.
361a /110b
721/230
771/247
800
962
1030/333
1038/336
3 4 5 3 4 5
c/T t/I c/T c/T c/T c/T
g/S g/S t/I g/S g/S g/S
a/T a/T g/A a/T a/T a/T
a/R a/R a/R a/R a/R t/R
c/Y c/Y t/Y c/Y c/Y c/Y
t/L t/L t/L c/P c/P c/P
g/V g/V g/V g/V g/V g/V
4 5 6 7
c/T c/T c/T c/T
t/I t/I t/I t/I
a/T a/T a/T a/T
a/R a/R a/R a/R
c/Y c/Y c/Y c/Y
t/L t/L t/L t/L
g/V a/I a/I a/I
c/T
g/S
a/T
a/R
c/Y
t/L
g/V
Input virus a b
Nucleotide position/amino acid position
Nucleotide positions. Amino acid positions.
ignations hereafter will be C for chicken and D for duck followed by a number for the individual bird) shed virus for 1 day while the other shed intermittently up to 13 days P.I. (C-7). Virus shedding among the contact chickens started on various days, from as early as day 1 (C-6) to as late as day 11 (C-2) after the inoculation of the index birds. Intermittent shedding was observed in all contact birds. In the chicken 50% BLP group, all naïve birds were infected by contact and the pattern of virus shedding was similar to that in the 20% BLP group. In the duck experiment, seven of eight naïve ducks became infected through contact (one duck died on the second day, unrelated to infection; Fig. 1). Both inoculated ducks (D-7 and D-10) started shedding the virus oropharyngeally on day 3 P.I. One duck (D-9), infected through contact, began shedding on day 4 P.I. No virus was detected from oropharyngeal or cloacal swabs collected from the negative control chickens or ducks. 3.2. Genetic and phenotypic adaptation Twenty-four isolates were recovered from eight infected chickens at various time points during the experiment. Five of the chickens were infected by inoculation and three by contact. Virus was not recovered from all of the infected chickens, presumably
Fig. 2. Fitted (lines) and observed (bars) frequency distribution of daily amino acid substitutions for chickens (triangles and shaded-striped bars) and ducks (circles and striped bars). The best fitting distribution is Poisson for ducks and negative binomial for chickens.
due to the low titers in those birds and intermittent shedding. Nucleotide sequences of HA and NA stalk regions of all 24 isolates were of the H11N9 subtype. A comparison of the HA genes between the recovered viruses and the inoculating virus identified five changes within the 1449 bases, one synonymous and four non-synonymous (numbering for both nucleotide and amino acid sequences are based on that of the H11 HA consensus; Table 1). The inoculating virus has Ala and Ser at positions 198 and 274 of HA protein, respectively, whereas 17 of the 24 recovered chicken isolates had Val198 and Phe274 instead (Table 1). Viruses from all contact chickens and two of the five inoculated chickens had Val198 and Phe274 (Table 1). One of the inoculated chickens (C-7) had two additional substitutions of Gly144Asp and Ser230Asn in the HA protein on day 8 P.I. Ten viruses were isolated from three infected Pekin ducks and mutations in the HA genes were detected in seven positions, five of which were non-synonymous (Table 2). One of the six substitutions (amino acid position 230) was common to the viruses recovered from one inoculated (D-7) and one contact (D-9) duck. Substitutions at amino acid positions 110, 247, 333 and 336 were unique for each individual isolates (Table 2). The number of daily nucleotide substitutions in the HA gene deviated significantly from a Poisson distribution for chickens (P = 0.02, likelihood ratio test) but not for ducks (Fig. 2). Viruses from two inoculated chickens (C-11 and C-12) had a 90-nucleotide deletion in the stalk region of the NA gene, representing 30 amino acid residues (positions 44–73, numbering of
Fig. 3. In vivo replication of the H11N9 viruses in inoculated chickens. , inoculating virus from C-5; , HA mutants from C-7 and C-14; , NA stalk deletion mutants from C-11 and C-12. Each point represents a 3-day rolling average titer.
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J. Li et al. / Virus Research 147 (2010) 40–46
Table 3 Serum conversion in chickens and Pekin ducks (HI unit). Chicken
Days post inoculation
C-5a C-7a C-11a C-12a C-14a Duck
D-3 D-7a D-8 D-9 D-10a
0
3
6
10
13
17
0 0 0 0 0
0 0 0 0 0
0 4 4 8 8
NT 8 32 2 2
4 32 16 0 0
4 0 16 0 0
Days post inoculation 0
5
7
11
14
18
0 0 0 0 0
0 0 0 0 0
0 0 0 0 16
0 0 4 16 32
0 0 2 4 16
4 4 NT 4 16
Fig. 4. Antigen-specific antibody against the inoculating H11N9 virus in Pekin ducks measured by plaque reduction assay. Numbers represent individual ducks. Asterisks indicate inoculated birds.
NT, not tested. a Inoculated birds.
A/duck/WA/663/97 NA protein) from the first day P.I. (Table 1). No NA stalk deletion was detected in the inoculating virus when tested by RT-PCR (data not shown). None of the seven viruses recovered from three chickens infected by contact had deletions in the NA stalk. No viruses were isolated from contact birds in the 50% BLP group from which NA stalk deleted viruses persisted for as long as 6 days. Viruses recovered from two infected and one contact duck had intact NA stalk regions. Viruses rescued from infected chickens could be divided into two groups based on their patterns of mutation. One group of viruses had amino acid substitutions in HA proteins (Table 1, C3, C-7, C-8, C-10 and C-14). Another group of viruses contained NA stalk deletions (Table 1, C-11 and C-12). The HA substitution viruses replicated to higher levels and for longer periods of time than the NA stalk deletion mutants in vivo, and both types of mutants replicated more efficiently than the inoculating virus (Fig. 3). 3.3. Host responses to virus infection The two inoculated chickens in the 20% BLP group (C-5 and C-7) and three in the 50% BLP group (C-11, C-12 and C-14) produced low levels of antibodies, ranging from 4 to 32 HI units against the inoculating virus whereas antibodies were not detected in any contact chickens (Table 3). Sera from the contact chickens did not neutralize the inoculating virus. One inoculated duck (D-10) displayed an antibody response on day 7 P.I. and produced the highest antibody titer of 32 HI units. In contrast to D-10, another inoculated duck, D7, had an antibody level of 4 HI units on day 18 P.I. A contact duck (D-9) from which we were able to isolate viruses, had antibody titer comparable to that of D-10. D-3 and D-8 had detectable antibodies in response to infection (Table 3), although no virus was recovered from them. In addition to the HI results, the plaque reduction assay (PRA) showed that the viruses elicited detectable humoral responses in Pekin ducks (Fig. 4). Sera from D-7, D-9 and D-10 neutralized more than 90% of the inoculating virus, which confirmed the HI results (Table 3). Sera from D-1, D-5, D-6 and D-8 neutralized more than 25%, but less than 80% of the inoculating virus. The detection of seroconversion by PRA and HI were not equivalent in these experiments. Sera from D-1, D-5 and D-6 were positive by PRA and negative for HI, while duck D-3 was negative by PRA and positive by HI (Table 3). Changes in HA protein during the course of infection may influence the antibody profile. A cross reactivity analysis was performed using virus and serum samples collected from two chickens at different time points. The viruses were from C-7 (inoculated chicken)
Fig. 5. Cross reactivity between viruses recovered from infected chickens and sera collected at various time points. (Legend denotes viruses isolated from chickens on specified days.)
on days 3, 4 and 8 P.I. and from C-8 (contact chicken) on day 5 P.I. (Fig. 5). Sera were collected from C-7 on days 6, 10 and 13 P.I. and C-8 on day 13 P.I. The antibodies from C-7 on day 6 P.I. only neutralized the inoculating virus but not viruses recovered at later days. Sera from C-7 at later time points neutralized all the viruses (Fig. 5). Serum collected from C-8 (contact chicken) on day 13 P.I. neutralized all tested viruses isolated 3 days after initial inoculation, but had no neutralizing activity against the inoculating virus. 4. Discussion There are a few AIV subtypes with no reported outbreaks in poultry, suggesting that subtype specific constraints may either prevent their infection of, or transmission between, domestic chickens and ducks. To explore this possibility, we used an H11N9 LPAIV of duck origin to infect two poultry species, chicken (Gallus gallus domesticus) and Pekin duck (Anas platyrhynchos domesticus). These studies showed that the original duck virus was transmissible among both hosts by direct contact. In both hosts, 100% of the inoculated and contact birds were infected during the study, as determined by either virus detection or seroconversion. Our findings demonstrated that genetic changes in the virus genes occurred at different positions and at different rates in chickens and in ducks. The pattern of nucleotide substitutions in HA genes of the duck isolates are consistent with a model that assumes substitutions appear independently of each other and at a constant rate (Rice, 1995). In contrast, nucleotide substitutions in HA genes among chicken isolates appeared to be non-random, suggesting
J. Li et al. / Virus Research 147 (2010) 40–46
specific selection pressures in chickens, presumably related to host adaptation. The genetic changes associated with the adaptation of an AIV to domestic avian hosts have been reported for other subtypes (Hossain et al., 2008), but not previously for H11N9. In these studies, we found that two amino acid substitutions in the HA protein (A198V and S274F) were consistent among viruses isolated from chickens. Viruses from two of the inoculated and all contact chickens had the same substitutions. Alanine and serine at these two positions from the inoculating virus were found common among all 64 currently published H11 HA proteins (McCracken, 2006). This suggests that chickens impose a selection regime on the H11 HA gene that is different from the selection processes in reservoir avian hosts. Amino acid changes in the HA proteins were more random and less persistent in the isolates from ducks compared to those from chickens (Tables 1 and 2). Additionally, the HA genes of the H11N9 virus in ducks had a continuous low rate of nucleotide substitutions, whereas, in chickens, changes occurred early and then remained throughout the experimental period. A possible explanation for this pattern is that the observed nucleotide substitutions in the H11 HA gene in ducks were results of random fluctuation in the frequency of different variants. In contrast, the selection by chickens for a particular virus variant reduces this fluctuation, and hence the observed lower daily substitution rate. We observed that the same synonymous substitution appeared together with two amino acid substitutions in the HA proteins of the viruses isolated from three inoculated chickens housed separately. Therefore, it is reasonable to assume that pre-existing quasi-species in the inoculum played an important role in the rapid adaptation of H11N9 to chickens. Alternatively, it is possible that rapid and frequent mutations could occur during the virus amplification processes in this host species. The stalk deletions in the NA proteins in viruses recovered from two inoculated chickens are not found in any of the 88 N9 NA sequences currently available in public databases (McCracken, 2006). There is no evidence to support the pre-existence of the stalk deletion in the inoculating virus after testing multiple batches of virus preparations with various dilutions using RT-PCR (data not shown), although NA stalk deleted virus below the detection limit of the test could have been present in the inoculum. We conclude that NA stalk deletion, which has been linked previously to the virus isolates from gallinaceous birds (Giannecchini et al., 2006), either developed rapidly in this AIV through new mutations or was rapidly selected. The appearance of an NA stalk deletion was not a frequent or consistent event in this study. Among 17 chickens inoculated with A/duck/WA/663/97 included in this report and a previous study (Li et al., 2008), only two birds (12%) shed viruses with NA stalk deletions. A comparison of in vivo replication kinetics between the NA deletion and HA substitution mutants in these studies demonstrated that the HA mutants replicated better and were transmitted by contact to other chickens. In contrast, the NA stalk deletion mutants replicated better in chickens than the inoculating virus, but less efficiently than the HA mutant viruses. Although it is likely that NA stalk deletion resulted in a virus more fit for growth in chickens, it clearly was not as critical as the HA substitutions. None of the amino acid substitutions in the H11 HA protein changed the pattern of glycosylation or receptor-binding sites. However, serological analysis suggested that the changes may have affected antibody neutralization (Fig. 5). One possible explanation is that virus adapting to new hosts undergoes continuous changes to evade antibodies, resulting in viruses that are less effectively neutralized during an infection. This is supported by the fact that viruses continued to replicate in the chickens after antibodies were detected, indicating an incomplete immune clearance. In addition to escaping humoral immune responses, mutations in the HA and
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NA proteins may also affect the efficiency of cell-mediated immune responses, which play a major role in AI pathogenesis (Swayne and Kapczynski, 2008). This study explored viral adaptation to poultry species by using a duck-origin isolate of a subtype rarely detected in domestic avian species. The data provided specific information on genetic changes and phenotypes of intermediate viruses isolated during adaptation. Based on these studies, it appears that there are few or no barriers to viral adaptation, even in subtypes that have not caused outbreaks in poultry. This provides critical testimony for the need for measures and practices that reduce contact between poultry and free-flying birds to prevent disease and to slow viral evolution. Acknowledgements Funding for these studies was provided by the Center for Food and Animal Health at the University of California, Davis. We would like to express our heartfelt appreciation to Dr. Zengqi Yang, Phuong Dao, Jerome Anunciacion, Heather Krabbenhoft, MaiLee Yang, and Kou Yang for their expert technical support. References Cecchinato, M., Bonfanti, L., Marangon, S., Terregino, C., Monne, I., Luppi, A., 2008. Low pathogenic avian influenza in Italy. Vet. Rec. 162 (2), 64. Chiang, C.L., 1980. An Introduction to Stochastic Processes and their Applications. Krieger Publishing Company, New York, USA. Choi, Y.K., Ozaki, H., Webby, R.J., Webster, R.G., Peiris, J.S., Poon, L., Butt, C., Leung, Y.H., Guan, Y., 2004. Continuing evolution of H9N2 influenza viruses in Southeastern China. J. Virol. 78 (16), 8609–8614. Fouchier, R.A., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B., Osterhaus, A.D., 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J. Virol. 79 (5), 2814–2822. Gaush, C.R., Smith, T.F., 1968. Replication and plaque assay of influenza virus in an established line of canine kidney cells. Appl. Microbiol. 16 (4), 588–594. Giannecchini, S., Campitelli, L., Calzoletti, L., De Marco, M.A., Azzi, A., Donatelli, I., 2006. Comparison of in vitro replication features of H7N3 influenza viruses from wild ducks and turkeys: potential implications for interspecies transmission. J. Gen. Virol. 87 (Pt 1), 171–175. Gill, J.S., Webby, R., Gilchrist, M.J., Gray, G.C., 2006. Avian influenza among waterfowl hunters and wildlife professionals. Emerg. Infect. Dis. 12 (8), 1284–1286. Hoffmann, E., Stech, J., Guan, Y., Webster, R.G., Perez, D.R., 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146 (12), 2275–2289. Hossain, M.J., Hickman, D., Perez, D.R., 2008. Evidence of expanded host range and mammalian-associated genetic changes in a duck H9N2 influenza virus following adaptation in quail and chickens. PLoS ONE 3 (9), e3170. Jahiel, R.I., Kilbourne, E.D., 1966. Reduction in plaque size and reduction in plaque number as differing indices of influenza virus-antibody reactions. J. Bacteriol. 92 (5), 1521–1534. Kawaoka, Y., Yamnikova, S., Chambers, T.M., Lvov, D.K., Webster, R.G., 1990. Molecular characterization of a new hemagglutinin, subtype H14, of influenza A virus. Virology 179 (2), 759–767. Keawcharoen, J., Oraveerakul, K., Kuiken, T., Fouchier, R.A., Amonsin, A., Payungporn, S., Noppornpanth, S., Wattanodorn, S., Theambooniers, A., Tantilertcharoen, R., Pattanarangsan, R., Arya, N., Ratanakorn, P., Osterhaus, D.M., Poovorawan, Y., 2004. Avian influenza H5N1 in tigers and leopards. Emerg. Infect. Dis. 10 (12), 2189–2191. Kinde, H., Read, D.H., Daft, B.M., Hammarlund, M., Moore, J., Uzal, F., Mukai, J., Woolcock, P., 2003. The occurrence of avian influenza A subtype H6N2 in commercial layer flocks in Southern California (2000-02): clinicopathologic findings. Avian Dis. 47 (3 Suppl.), 1214–1218. Lamb, R.A., 1989. Genes and proteins of the influenza viruses. In: Krug, R.M., Fraenkel-Conrat, H., Wagner, R.R. (Eds.), The Influenza Viruses. Plenum Press, New York, pp. 1–88. Li, J., Cardona, C.J., Xing, Z., Woolcock, P.R., 2008. Genetic and phenotypic characterization of a low-pathogenicity avian influenza H11N9 virus. Arch. Virol. 153 (10), 1899–1908. Londt, B.Z., Nunez, A., Banks, J., Nili, H., Johnson, L.K., Alexander, D.J., 2008. Pathogenesis of highly pathogenic avian influenza A/turkey/Turkey/1/2005 H5N1 in Pekin ducks (Anas platyrhynchos) infected experimentally. Avian Pathol. 37 (6), 619–627. McCracken, C., 2006. The Influenza Sequence Database. Los Alamos National Laboratory. Nguyen, D.C., Uyeki, T.M., Jadhao, S., Maines, T., Shaw, M., Matsuoka, Y., Smith, C., Rowe, T., Lu, X., Hall, H., Xu, X., Balish, A., Klimov, A., Tumpey, T.M., Swayne, D.E., Huynh, L.P., Nghiem, H.K., Nguyen, H.H., Hoang, L.T., Cox, N.J., Katz, J.M., 2005. Isolation and characterization of avian influenza viruses, including highly
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