Department of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy1
Avian Influenza Reference Centre, University Federico II, 80014 Naples, Italy2
Istituto Zooprofilattico Sperimentale, 43100 Parma, Italy3
Istituto Nazionale della Fauna Selvatica, 40064 Ozzano Emilia, Bologna, Italy4
Department of Virology and Molecular Biology, St Jude Childrens Research Hospital, Memphis, TN 38105-2794, USA5
Author for correspondence: Laura Campitelli. Fax +39 06 49 902 082. e-mail campitel{at}iss.it
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Abstract |
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Introduction |
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In European pigs, H3N2 influenza viruses possess genes encoding non-surface proteins of avian origin (Campitelli et al., 1997 ). This phenomenon was caused by a reassortment event between human-like H3N2 swine influenza viruses and avian-like H1N1 swine influenza viruses (Castrucci et al., 1993
). These viruses, which have been circulating during the past 15 years, can be transmitted to and cause disease in humans (Claas et al., 1994
).
During the winter of 19941995, we monitored several chicken farms in northern Italy for the presence of respiratory pathogens in commercial poultry showing signs of mild respiratory disease and a temporary reduction in egg production. Three H3N2 influenza virus strains were isolated from broiler chickens and battery hens located in three distinct farms. Because the farms containing chickens from which H3N2 influenza viruses were isolated were located in an area with many pig farms and because H3N2 swine influenza virus had been detected in those pigs during the autumn of 1994, we questioned whether the pigs were the source of the H3N2 influenza virus that infected the chickens.
Here we report the ability of H3N2 influenza viruses from Italian poultry to replicate in the respiratory tract of experimentally infected chickens and the antigenic and molecular relationships between H3N2 influenza viruses of chickens and swine in Italy.
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Methods |
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Antigenic characterization.
Haemagglutination inhibition (HI) assays were performed according to standard procedures (US Department of Health and Human Services, 1982 ). Viruses were tested with a panel of hyperimmune polyclonal antisera to the following viruses: Dk/Ukr/1/63 (H3N8), Aichi/1/68 (H3N2), PChal/1/73 (H3N2) and Coot/It/153/94 (H3N8), a recent wild duck isolate. Monoclonal antibodies (mAbs) to the early human H3N2 influenza virus strains England/42/72 and Tex/1/77 were also used.
Experimental infections.
Four groups of 3-week-old SPF White Leghorn chickens (n=4 birds per group) were inoculated intranasally, orally and intratracheally with one of the four avian influenza viruses. Tracheal and cloacal swabs were collected 3 days after infection. Swabs and faeces were suspended in PBS with antibiotics and the suspensions were injected into 11-day-old embryonated chickens eggs for isolation and titration of virus.
To evaluate the extent of replication and shedding of the H3N2 influenza virus chicken isolates, five 3-week-old SPF White Leghorn chickens were inoculated intranasally, orally and intratracheally with allantoic fluid containing Ck/It/5945/95 (H3N2) influenza virus. Tracheal swabs were collected on days 15 after infection for isolation and titration of virus. Two birds were dissected on day 3 to determine the organs in which the virus was replicating. Tissue samples of brain, lung, trachea, colon, spleen, heart, blood, gall bladder, pancreas and intestine were collected aseptically for virus titration. Suspensions (10% v/v) were prepared in a Stomacher blender and the virus was assayed as described previously (Webster et al., 1981 ).
To evaluate the degree of pathogenicity of the viruses in chickens, we infected chickens intravenously with one of the three swine influenza virus strains isolated during different years (Sw/It/526/85, Sw/It/25823/94 or Sw/It/1452/96) or with the human variant PChal/1/73. Except for Sw/It/25823/94, which was tested in four birds, each virus was tested in two birds.
DNA sequence analysis.
We used the RNeasy kit (Qiagen) to extract viral RNA (vRNA) from infected allantoic fluid and RTPCR to amplify gene segments, as described previously (Lin et al., 1994 ). Briefly, cDNA was synthesized using reverse transcriptase and a 12 bp oligodeoxynucleotide primer complementary to a common sequence in the 3' terminus of all influenza vRNA genes. Regions of gene segments encoding internal proteins were amplified using gene-specific primers. PCR products were sequenced using the Prism Ready Reaction Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems) and an ABI 373A automated sequencer (Applied Biosystems).
Phylogenetic analysis.
We used the GCG program package, version 8.0, to assemble a map of contiguous nucleotides and to perform multiple sequence alignments. Evolutionary analysis of the gene segments was performed by using the neighbour-joining method of the PHYLIP software package, version 3.5 (Felsenstein, 1993 ). Gene sequences available from GenBank were used in all sequence comparisons and phylogenetic analyses.
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Results |
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Three H3N2 influenza viruses were isolated from samples taken at three different farms, which raised a total of 103000 birds. Ck/It/5954/95 and Ck/It/5945/95 influenza viruses were present in tracheal samples, whereas Ck/It/5783/95 influenza virus was found in ovary samples.
During the weeks before the influenza outbreak in chickens, the H3N2 swine influenza virus Sw/It/25823/94 had been isolated from a nearby swine herd that belonged to the same commercial network. Farm personnel had worked at both the swine farm and the poultry farms.
Pathogenicity of H3N2 chicken and swine influenza virus isolates in domestic chickens
Because the chicken and swine H3N2 influenza virus strains were isolated in temporal and geographical proximity in Italy, we hypothesized that the avian strains may have been derived from a virus infecting a swine host. To test this hypothesis, we compared first the pathogenicity of the two virus groups in experimentally infected chickens. When chickens were inoculated intranasally, orally and intratracheally, the three chicken strains (Ck/It/5783/95, Ck/It/5954/95 and Ck/It/5945/95) replicated in the oropharyngeal tracts of the infected birds but no virus was recovered from the cloacae (Table 1). The birds showed no signs of disease and their consumption of food did not decrease during the study. The three H3N2 swine influenza virus strains (Sw/It/25823/94, Sw/It/1452/96 and Sw/It/526/85) and the antigenically related early human influenza virus PChal/1/73 did not replicate in either the respiratory tract or the intestinal tract of chickens. Antibody levels in sera of chickens that were experimentally infected with the avian strains were consistently elevated 10 days after infection, whereas antibody levels in sera of chickens infected with the human and swine strains were comparable to the low non-specific antibody titres in sera before infection (data not shown).
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Analysis with polyclonal antisera showed antigenic cross-reactivity among the swine and chicken H3N2 influenza virus isolates from Italy, with highest titres to Aichi/1/68 and PChal/1/73 (Table 3). The antisera for Aichi/1/68 reacted weakly with Coot/It/153/94 and the Dk/Ukr/63 and PChal/1/73 antisera did not react with Coot/It/153/94, an H3N8 influenza virus strain isolated from wild waterfowl in central Italy in 1994. In contrast, the Coot/It/153/94-specific antiserum, which reacted with two early human influenza virus variants (PChal/1/73 and Tex/1/77), also reacted with Sw/It/526/85. This finding illustrates the reduced sensitivity of the HI antibody to avian viruses, as reported previously (Lu et al., 1982
).
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Sequence and phylogenetic analysis of the genes encoding HA and internal proteins
To evaluate the genetic relationships among the swine and chicken H3N2 influenza viruses and to elucidate the origin of the chicken H3N2 influenza virus strains, we sequenced portions of genes encoding HA1 and the six internal proteins of the chicken H3N2 influenza viruses in Italy and compared these nucleotide sequences with corresponding sequences from other avian and swine influenza virus strains.
Genes encoding HA1.
Sequence similarity among the genes of the three chicken viruses ranged from 99·5 to 100% (data not shown). Of the HA1 genes of H3 influenza viruses from different animal hosts, the HA1 genes of duck influenza viruses of Eurasian origin were most similar to those of the representative chicken influenza virus Ck/It/5945/95 (88·2%) (Table 4). On the other hand, similarity between the HA1 gene of Ck/It/5945/95 and Sw/It/25823/94 was much lower (82·1%). The swine strain Sw/It/25823/94 was related most closely to H3N2 swine influenza viruses isolated recently from northern Europe (97·9% similarity). Sw/It/25823/94 showed a marked genetic drift from the earlier Italian H3N2 influenza virus isolates Sw/It/309/83, Sw/It/520/85 and Sw/It/635/87 (between 92·4 and 92·6% similarity; data not shown).
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Phylogenetic analysis of the NP gene (Fig. 2) identified distinct subgroups within the Eurasian avian influenza virus lineage. The NP gene of the Italian avian H3N2 influenza viruses clustered with that of Sw/HK/168/93, an H1N1 influenza virus of avian origin that was detected recently in pigs in China (Guan et al., 1996
). In contrast, the NP gene of the H3N2 swine influenza virus clustered with those of the European swine influenza viruses of avian descent. Both branches were distinguishable from the group represented by the NP gene of HK/156/97. This group also included a strain isolated from wild ducks in Italy (Mal/It/24/95; H1N1) (L. Campitelli, unpublished data). Thus, on the basis of the antigenic and phylogenetic data, the chicken influenza viruses do not seem to be derived from contemporary strains circulating in wild ducks in Italy, such as Coot/It/153/94 and Mal/It/24/95. Instead, the antigenic heterogeneity among the avian H3 proteins and the genetic diversity among the NP genes indicate that multiple sublineages exist within the gene pool of avian influenza viruses that are circulating in Italy.
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Discussion |
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Because human H3N2 influenza viruses that infected pigs in Italy before 1980 continue to circulate (Castrucci et al., 1994 ; Campitelli et al., 1997
) and were found shortly before the avian virus isolations in pigs on farms near those where the outbreak in chickens occurred, we hypothesized that the Italian H3N2 chicken influenza virus arose from strains that were present in the nearby pigs (Ludwig et al., 1994
). However, results of our antigenic analysis with mAbs and results of our sequencing analysis of the genomes of the avian H3N2 influenza viruses do not support this hypothesis. Instead, the chicken H3N2 influenza viruses in Italy were most similar to the Eurasian avian influenza viruses. HA sequence and evolutionary analyses suggest an introduction of the virus from wild aquatic birds, although the relatively low percentage of similarity (88·2%) between the HA1 gene of Ck/It/5945/95 and that of Dk/Ho/5/77 indicates that the Italian chicken influenza viruses are only distantly related to other known avian H3 influenza virus strains. In the NP phylogenetic tree, the viruses under study were grouped with an avian-like swine influenza virus from Hong Kong (Sw/HK/168/93; H1N1).
Several different subtypes of influenza viruses have been isolated from domestic chickens. The two highly pathogenic subtypes H5 and H7 have been isolated from chickens for many years (Stubbs, 1965 ; Bean et al., 1985
). Other influenza viruses isolated in chickens include the following subtypes: H1 (Siebinga & de Boer, 1988
), H2 (Schäfer et al., 1993
), H4 (Donis et al., 1989
), H6 (Lin et al., 1994
) and H10 (Feldmann et al., 1988
). Unlike the highly pathogenic strains, these less pathogenic viruses have been detected sporadically and appear to be confined to the poultry species from which they had been isolated.
The transmission of H9N2 influenza viruses to domestic chickens in Asia, the establishment of stable lineages of influenza virus strains in chickens (Guan et al., 1999 ; Guo et al., 2000
) and the transmission of these viruses to humans (Peiris et al., 1999
) suggest that the role of domestic chickens in the ecology of influenza viruses may be expanding. Alternatively, these three observations may result from increased surveillance, which is more frequently detecting viruses that are transmitted to other species but do not necessarily establish themselves in that species. As domestic chickens become an important source of protein for humans, their increasing population raises the possibility of an increased role for them in the ecology of influenza.
Our studies suggest that the circulation of non-pathogenic influenza viruses in commercial poultry may be significantly underestimated because these infections are asymptomatic and are often not detected. This theory raises some concern because of the episodes of transmission of highly pathogenic H5N1 chicken influenza viruses to humans in Hong Kong (de Jong et al., 1997 ; Claas et al., 1998
; Subbarao et al., 1998
; Suarez et al., 1998
). Although the genetic determinants that favour interspecies transmission of avian influenza viruses have not been identified, the role of the host cell receptor does not appear to be crucial. Receptor specificity for avian viruses, as defined by oligosaccharides that contain the Sia(
2,3)Gal-linkage (Rogers & Paulson, 1983
), does not necessarily restrict avian-to-human transmission (Matrosovich et al., 1999
). This finding suggests that chickens may also serve as an intermediate host either instead of or in addition to the pig. The presence of Sia(
2,3)-containing (avian influenza virus-specific) receptors and Sia(
2,6)-containing (human influenza virus-specific) receptors (Ito et al., 1998
) in pigs makes this an attractive model for transmission of avian influenza viruses to man, but the direct transmission of H5N1, H9N2 and H7N7 (Kurtz et al., 1996
) avian influenza viruses to humans indicates that the interspecies transmission of influenza virus does not necessarily require the pig.
The antigenic differences among the H3N2 influenza viruses isolated from chickens in Italy and the H3N8 influenza viruses from wild aquatic birds of the same region suggest that multiple types of H3 influenza viruses are circulating in Italy. This finding also indicates that an extensive gene pool for influenza viruses continues to circulate in nature; however, the actual source of the H3N2 influenza virus that infected chickens in Italy is unknown. Therefore, our studies emphasize the continued need to monitor domestic and wild avian populations to better understand interspecies transmission and the importance of avian hosts in the ecology of influenza.
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Acknowledgments |
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Footnotes |
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References |
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Received 2 August 2001;
accepted 2 November 2001.