Pandemic Potential of Reassortant Swine Influenza A Viruses

Influenza A viruses are capable of causing disease in several species, including birds, humans and swine. Host specificity of the viruses is not absolute, and is influenced by a range of factors. Swine play a pivotal role in the interspecies transmission of influenza A viruses, as they are susceptible to infection with both human and avian strains and have been implicated as a “mixing vessel” for the reassortment of influenza A viruses from different species. The reassortment of influenza A viruses of human and avian origin led to human influenza pandemics in 1957 and 1968. The dynamics of swine influenza viruses in North America changed drastically with the introduction of the avian-origin PA and PB2 and human-origin HA, NA, and PB1 gene segments and the creation of the triple reassortant swine virus lineage in 1998. While the previously circulating classical swine H1N1 influenza virus lineage was very stable in the swine population, triple reassortant lineage viruses have supplanted the classical H1N1 lineage and undergone repeated reassortment events, acquiring HA and NA genes from human, swine, and avian influenza viruses, while maintaining triple reassortant internal gene (TRIG) cassette. Viruses of the triple reassortant lineage have been very successful in the swine population, yet the mechanisms underlying their unique characteristics and increased fitness have not been elucidated. Here we address the pandemic potential of triple reassortant swine influenza A viruses, their transmissibility, and their relative fitness compared to classical and double reassortant swine influenza viruses. Several triple reassortant viruses, including one with avian-origin HA and NA, were characterized in the ferret, which is a commonly used model for human influenza infection. The effect of the TRIG cassette on the reassortment potential and temperature sensitivity of swine influenza viruses was determined in cell culture, and the replication and transmission of a classical and a reassortant swine virus were compared in pigs. We found that triple reassortant swine viruses replicated efficiently in the ferret model, although there was some variation in transmission efficiencies. An H2N3 virus with avian-origin HA and NA was transmissible in the ferret model, and this transmissibility could be abolished with a single amino acid change in the HA protein that altered its receptor binding specificity. Avian H2N3 viruses were also capable of replicating in ferrets without adaptation and could acquire transmissibility through a change in the receptor binding specificity of the HA protein. Both double and triple reassortant swine viruses had an advantage over the classical H1N1 swine virus at early timepoints in cell culture. Reassortant viruses also demonstrated less temperature sensitivity than the classical H1N1 swine virus. The triple reassortant H1N1 virus had an increased reassortment potential in cell culture compared to the classical swine H1N1 virus as determined by acquisition of a human HA gene. Triple reassortant swine viruses have an increased ability to establish infection, and an increased potential for reassortment, potentially introducing novel HA genes into a host population. This indicates that triple reassortant swine viruses may have an increased potential to cause human pandemics. In April 2009, a novel H1N1 pandemic virus containing five of the six genes of the TRIG cassette emerged in the human population, emphasizing the importance of reassortant swine influenza A viruses in the generation of human pandemics

Replication and Transmission of H9N2 Influenza Viruses in Ferrets: Evaluation of Pandemic Potential

H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT) H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu) residue at amino acid position 226 in the hemagglutinin (HA) receptor-binding site (RBS), responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans

Multiple reassortment between pandemic (H1N1) 2009 and endemic influenza viruses in pigs, United States.

As a result of human-to-pig transmission, pandemic influenza A (H1N1) 2009 virus was detected in pigs soon after it emerged in humans. In the United States, this transmission was quickly followed by multiple reassortment between the pandemic virus and endemic swine viruses. Nine reassortant viruses representing 7 genotypes were detected in commercial pig farms in the United States. Field observations suggested that the newly described reassortant viruses did not differ substantially from pandemic (H1N1) 2009 or endemic strains in their ability to cause disease. Comparable growth properties of reassortant and endemic viruses in vitro supported these observations; similarly, a representative reassortant virus replicated in ferrets to the same extent as did pandemic (H1N1) 2009 and endemic swine virus. These novel reassortant viruses highlight the increasing complexity of influenza viruses within pig populations and the frequency at which viral diversification occurs in this ecologically important viral reservoir.As a result of human-to-pig transmission, pandemic influenza A (H1N1) 2009 virus was detected in pigs soon after it emerged in humans. In the United States, this transmission was quickly followed by multiple reassortment between the pandemic virus and endemic swine viruses. Nine reassortant viruses representing 7 genotypes were detected in commercial pig farms in the United States. Field observations suggested that the newly described reassortant viruses did not differ substantially from pandemic (H1N1) 2009 or endemic strains in their ability to cause disease. Comparable growth properties of reassortant and endemic viruses in vitro supported these observations; similarly, a representative reassortant virus replicated in ferrets to the same extent as did pandemic (H1N1) 2009 and endemic swine virus. These novel reassortant viruses highlight the increasing complexity of influenza viruses within pig populations and the frequency at which viral diversification occurs in this ecologically important viral reservoir

Clinical signs, virus replication and seroconversion in inoculated ferrets.

a<p>Average body weight loss±SD is shown.</p>b<p>Homologous virus was used in the HI assays to detect anti-H9 antibodies.</p>c<p>For the RGWF10 virus, two separate experiments were performed.</p>d<p>Only one ferret lost body weight.</p

Replication and transmission of the H9N2 avian-human reassortant virus.

<p>Ferrets were inoculated i.n. with 10⁶ TCID₅₀ of 2WF10:6M98 virus. Twenty-four hours later, the direct and aerosol contacts were placed in the cages as described above. Nasal washes were collected daily and were titrated by TCID₅₀. (A) 2WF10:6M98 virus inoculated and direct contacts. (B) 2WF10:6M98 virus aerosol contacts.</p

Wild-type influenza viruses used in this study

a<p>RGWF10, RGQa88 and RGMemphis98 are WT viruses generated by reverse genetics.</p

Replication and transmission of mutant H9N2 viruses.

<p>Ferrets were inoculated i.n. with 10⁶ TCID₅₀ of mWF10 (Gln226) or mQa88 (Leu226) virus. Twenty-four hours later, contact ferrets were introduced as described above. Nasal washes were collected daily and were titrated by TCID₅₀. (A) mWF10 infected and direct contacts. (B) mWF10 aerosol contacts. (C) mQa88 infected and direct contacts. <i>L</i> and <i>Q</i> correspond to Leu226 and Gln226, respectively in the HA RBS.</p

Recovery and plaque assay of an H9N2 avian-human reassortant virus.

<p>(A) Diagram outlining gene segment exchange to create the reassortant virus. (B) Plaque morphology of the parental H3N2 virus RGMemphis98 (left), the parental H9N2 virus RGWF10 (center) and the 2WF10:6M98 reassortant virus (right).</p

Replication and direct contact transmission of H9N2 viruses.

<p>Ferrets were inoculated intranasally (i.n.) with 10⁶ TCID₅₀ of H9N2 viruses RGWF10 (A), Dk/HK/Y280/97 (B), RGQa88 (C), Ck/HK/SF3/99 (D), and Ck/Jordan/554/03 (E). Twenty-four hours later, one naïve ferret (direct contact) was added to the same cage as each of the infected ferrets. Nasal washes were collected daily and were titrated in MDCK cells. Black, white and gray bars represent individual ferrets sampled and the amount of viral shedding at different days pi. The titers are expressed as log₁₀ values of TCID₅₀/ml with the limit of detection at 0.699 log₁₀TCID₅₀/ml. The dotted line was arbitrarily set at <0.3 log₁₀TCID₅₀/ml in order to represent samples below the detection limit. <i>L</i> and <i>Q</i> correspond to Leu226 and Gln226, respectively in the HA RBS.</p