Influenza A viruses are highly contagious pathogens that have been isolated form a wide variety of animals, including man, birds, swine, horses, minks, seals, whales, and most recently from cats and dogs [1–3]. Influenza A viruses are rarely known to cross species barriers [4, 5], however, their interspecies transmission has always been a major concern. Although determinants of interspecies transmission are still not fully identified, many studies showed that the compatibility between the hemagglutinin (HA) protein of the virus and its corresponding receptor on the host cell is essential for establishing an infection in a specific host [6–8]. Pigs are known to be a major reservoir for H1N1 and H3N2 influenza viruses and have been hypothesized to act as intermediate host for interspecies transmission of influenza A viruses [6, 9, 10]. Turkeys on the other hand, are susceptible to a wide range of influenza A viruses and serve as an important host for these viruses [11, 12]. Influenza infections in turkeys range from asymptomatic to severe disease, including respiratory tract disorder, depression, drop in eggs production and high mortality [11]. Between 1978 and 1981, our laboratory was the first to report on experimental and natural infections of turkeys with H1N1 swine influenza viruses [13, 14].

In 1998, a new lineage of swine influenza viruses, triple reassortants (TR) H3N2, were isolated for the first time from pigs in the United States (U.S.) [15]. These viruses had genes derived from human (HA, NA, and PB1), Swine (NP, M, and NS) and avian viruses (PA and PB2) [16, 17]. The H3N2 TR viruses are now endemic in swine populations in North America [17, 18]. In 2003 and 2004, similar viruses (H3N2 TR) were isolated from turkeys in two different locations in the U.S. [19, 20]. Later in the same year, we isolated another H3N2 TR virus from turkey breeder hens in Illinois that were vaccinated twice with a swine H3N2 TR virus. Infected turkeys experienced complete cessation of egg production, but had no other clinical signs. In a previous study (manuscript submitted) we observed major antigenic differences between turkey and swine H3N2 TR viruses. The antigenic relatedness (R-value) between the turkey viruses and the swine virus (vaccine strain) was less than 30% as expressed by the Archetti and Horsfall formula [21] based on hemagglutinin inhibition (HI) and virus neutralization (VN) tests. At least eight amino acid changes were observed at the antigenic sites of the HA1 molecule between the turkey viruses and the swine vaccine virus. Although the transmission of H3N2 TR viruses from pigs to turkeys was suggested in previous reports [19, 20], no experimental work has been done to support this premise. Hence, we initiated this study to evaluate the interspecies transmission of these viruses between swine and turkeys, and to determine at the molecular level the basis for such transmission. Additionally, we tested one strain, A/turkey/Ohio/313053/04, that was shown to transmit between swine and turkeys for its intraspecies transmission in turkeys, chickens and ducks. Identifying viruses with different transmission potential between swine and turkeys will help in identifying the molecular determinants that control such transmission using the reverse genetics techniques.

Generally, influenza A viruses are considered host specific, nevertheless, some can overcome the species barrier and infect a new host. The mechanisms by which the influenza A viruses cross the species barriers and the molecular determinants that control such transmission are not well identified. Pigs have been hypothesized to play a role in interspecies transmission by acting as "mixing vessel" for the generation of reassortant viruses that might have the potential to jump from one species to another [22, 23]. In 1998, a new lineage of swine viruses, H3N2 TR, emerged and caused influenza like illnesses in pig populations in the U.S. [15, 16]. Similar viruses were later isolated from turkey breeder hens experiencing drop in eggs production and it was hypothesized that these viruses were transmitted from pigs to turkeys [19, 20].

Our findings indicated the ability of certain H3N2 TR viruses to transmit between pigs and turkeys. Despite the high degree of molecular similarity between some of these viruses, like A/turkey/Illinois/04 and A/turkey/Ohio/313053/04 (>99% similarity in all genes), they behaved differently in the transmission experiments, with the A/turkey/Ohio/313053/04 transmitting both ways between the two species and the A/turkey/Illinois/04 virus not transmitting either way.

Regardless of the differences in transmission, all viruses were capable of replication in turkeys and pigs but to different titers. Furthermore, the A/turkey/Ohio/313053/04, the strain transmissible between pigs and turkeys, was shown to infect and transmit among turkeys, infect but did not transmit among chickens, and did not infect ducks.

We speculate that the H3N2 TR viruses, which have the HA gene from human lineage viruses, retain the receptor binding specificity to NeuAcα2,6Gal receptors similar to human influenza viruses. Val226 and Ser228 were expressed in the HA1 molecules of both turkey and swine triple reassortants, while Leu/Ile226 and Ser228 are usually expressed in the human viruses [24]. Leu, Ile, and Val are neutral non-polar amino acids, and substitutions between them most likely maintain the hydrophobic interactions and the proper conformation at the binding domain [25]. Gln226 and Gly228 are usually found in the HA1 molecules of avian viruses amino acids at these positions and are known to play a critical role in determining the receptor binding specificity [25]. Our unpublished work demonstrated the presence of substantial amount of NeuAcα2,6Gal receptors in turkey tracheas, which would explain the ability of these viruses to replicate in turkeys as well as in pigs that are known to express these receptors [6]. Although ducks were shown to express few NeuAcα2,6Gal receptors in their tracheas (unpublished work), the A/turkey/Ohio/313053/04 H3N2 virus was not able to replicate in ducks. The absence of a large number of NeuAcα2,6Gal receptors in ducks' tracheas may explain the inability of the A/turkey/Ohio/313053/04 H3N2 virus to replicate in ducks. However, there may be factors other than receptors distribution that contribute to host tropism of influenza viruses and more work is needed in this area.

While all viruses had the Asp (D) amino acid at residue 190 of the receptor binding domain (RBD), a D to A (Ala) change occurred upon the transmission of the A/turkey/Ohio/313053/04 virus from turkeys to pigs. The presence of either D (specific for SAα2,6- gal) or E (specific for SAα2,3- gal) at amino acid position 190 of the HA molecule in the H3 subtypes was reported in previous studies [26, 27], however, our observation of (A) residue at this position is the first of its kind to our knowledge (sequencing was performed on the HA gene of the Ohio virus isolated from three different pigs in contact with infected turkeys). The role of (A) residue at position 190 in determining receptor binding specificity should be further investigated. In addition, the role of Asn (N) residue at position 246 of the HA molecule is not known and will be further studied in out laboratory.

Although all viruses were shed by pigs for more than 6 days, the A/turkey/Ohio/313053/04 and A/turkey/North Carolina/03 viruses replicated to higher titers than A/turkey/Illinois/04 and A/swine/North Carolina/03 viruses. This might be one of the possible reasons that allowed A/turkey/Ohio/313053/04 and A/turkey/North Carolina/03 viruses to transmit from pigs to turkeys (all animals were inoculated with the same virus titer). The A/turkey/Illinois/04 and A/swine/North Carolina/03 viruses were detected only on one day in contact turkeys by RRT-PCR, however, no viruses were obtained upon isolation attempts. The high sensitivity of the RRT-PCR might explain the ability to detect these viruses in contact turkeys, whereas the viruses were inefficient in replicating to a high titer in turkeys. In contrast, the A/turkey/Ohio/1988 H1N1 virus was shown to replicate to a very high titer in pigs (10^7.1 TCID₅₀), but it did not transmit to turkeys. The above observations indicate the specificity of individual influenza A viruses, even within the same subtype (H3N2 TR in this case), in their ability to transmit between species.

Previous analysis of the A/swine/North Carolina/03 virus in our laboratory showed that it has a 13 amino acids stalk deletion in the NA protein (manuscript submitted). Shortened NA stalks might result in less efficient virus release, and hence lower virus titers [28, 29]. This might explain our results from pigs and turkeys. However, the exact effect of NA stalk deletion is not clear because many chicken adapted H5, H7, and H9 viruses show different length stalk deletions and replicate to very high titer in poultry [30–32].

The identification of viruses with varying potential for interspecies transmission should be useful for reverse genetic studies to identify the gene(s) and the amino acid(s) residues that contribute to the transmission of these viruses between swine and turkeys. The use of the reverse genetics and site directed mutagenesis could also be helpful in deciphering the role of residues 190 and 246 of the HA molecule in receptor binding specificity and transmission of these viruses between swine and turkeys.

Interspecies transmission studies between swine (mammalian) and turkeys (avian) will enhance our understanding of the genetic factors that control transmission of influenza viruses and would help in improvement of surveillance strategies for early detection of influenza A viruses.