In recorded human history, H1N1 influenza viruses have been a major cause of human pandemics. Because these viruses have successfully established themselves in the human, swine, and avian populations, there is substantial risk of the gene pools of viruses from different host origins mixing. An influenza A pandemic occurred in 2009, and its genetic constellation of human, swine, and avian origin 
again highlighted the potential risk of H1N1 viruses and the importance of the gene pool in the wild bird population. In this study, we determined the pathogenicity of a group of representative avian H1N1 viruses of North American lineage in the mouse model. We further evaluated a few selected viruses in a ferret model to determine the pathogenicity and transmissibility of avian H1N1 viruses.
Although the severity of disease caused by the H1N1 viruses varied in the mouse model, 10 of 31 isolates were placed in the most pathogenic (PI-4) category. Of those 10 viruses, 8 were as pathogenic or more pathogenic than the pandemic A/TN/1-560/2009 virus, while the pathogenicity scores of 2 isolates were almost identical to that of the pandemic A/CA/04/2009 virus in DBA/2J mice. Thus, some avian H1N1 viruses are more pathogenic than the 2009 pandemic strains in the DBA/2J mouse model.
The pandemic A/CA/04/2009 virus requires as many as 9 lung-to-lung passages to be lethal in BALB/cJ mice 
. The 2 pandemic strains used in this study were also much less pathogenic (PI-0 to PI-1) in BALB/cJ mice. Considering BALB/cJ mice are more resistant to influenza A infection than are DBA/2J mice, the fact that the A/mallard/MN/AI07-3136/2007 isolate with 80% morbidity was more pathogenic than A/CA/04/2009 or A/TN/1-560/2009 in BALB/cJ mice without mouse adaptation raises concern about the avian H1N1 viruses residing in wild bird populations. Overall, our findings indicate that some avian H1N1 viruses in the wild bird reservoir could be more virulent than the 2009 pandemic strains in mammals and could be a potential concern for veterinary and human public health.
The main goal of this study was to evaluate the disease potential of low-pathogenic avian H1N1 influenza viruses in mammalian models, but we also tested other subtypes of low-pathogenic avian influenza A viruses to compare their pathogenicity levels. We selected the viruses from different categories: the ones detected in humans (H2, H3), those of avian origin but previously detected in mammals (H4, H6, H10), and those of purely avian origin and not yet detected in mammals (H12). In many instances, the DBA/2J mice either did not show any symptoms or recovered from the disease within a few days. The H10N4 and H10N7 isolates that caused mortality in DBA/2J mice were previously characterized in minks 
. Therefore, the higher pathogenicity of these viruses in a mouse model can be possibly explained by the tendency of H10N4 and H10N7 viruses to cross the species barrier under certain circumstances. H6 viruses are infectious to mice and ferrets 
. We found that 1 isolate, A/shorebird/DE/124/2001 (H6N2), was pathogenic in the mouse model. Likewise, H4N6 viruses have been isolated from pigs 
. Our results confirm the potential pathogenicity of H4N6 avian influenza A viruses in mammals. None of the H12 isolates used in this study caused disease signs in the mouse model, thereby confirming the absence of H12 viruses in mammals so far. Although several isolates were variably virulent in the DBA/2J mice, none were characterized in the PI-4 category. These findings suggest that H1N1 influenza A viruses of avian origin are prone to be more pathogenic in mammals than are many other avian influenza A viruses.
The pathogenicity and transmissibility of the 2009 pandemic H1N1 viruses via direct or respiratory-droplet contact was previously reported in a ferret model 
. Our results indicate that 2 avian H1N1 viruses used in this study caused infection in donor ferrets, as well as in ferrets in direct contact with those donors. Furthermore, the A/shorebird/DE/300/2009 isolate successfully transmitted to respiratory-droplet naïve-contact ferrets, as was seen for the pandemic 2009 H1N1 strains.
The ferret model is the best mammalian model widely used in influenza research, because it closely mimics the progression, pathogenicity, and transmissibility of influenza A infections in humans. However, this animal model is high maintenance, i.e., ferrets are more expensive; they require more space per animal, and their care is labor-intensive. These factors are a big obstacle for a broad experimental setup to assess large numbers of avian viruses. Although mice are not naturally infected with influenza viruses, and inbred strains lack the Mx gene to inhibit viral replication 
, mice are still widely used because they are a low-maintenance animal model (i.e., more reagents and serum are available for vaccine and antiviral assays; mice also cost less, require less space per animal, and are less labor-intensive). Although BALB/cJ mice have been a broadly preferred model, recent studies have shown that DBA/2J mice are more sensitive to influenza A infection than BALB/cJ 
or C57BL/6 mice 
. High virulence of avian H1N1 viruses in the DBA/2J confirms that this strain is a suitable model in which to conduct initial pathogenicity screens of large numbers of viruses. Moreover, varying degrees of pathogenicity and transmissibility of 2 selected avian H1N1 viruses from 2 separate pathogenicity levels in a ferret model confirm the PI values assigned in the DBA/2J model. Further assessment of several more avian H1N1 viruses from the PI-4 category and the representative virus isolates from other pathogenicity levels in the ferret model would support DBA/2J mice as a suitable mammalian model for the initial screening of vast quantities of avian H1N1 viruses from the wild bird repository. This is an important first step in establishing an efficient, long-term risk-assessment and risk-management program for H1N1 influenza A viruses of avian origin.
Influenza A viruses have been previously detected in the intestines of ferrets and pigs 
. The viral titers we detected in the intestines of some mice on 5 dpi were consistent with those earlier findings. During the 2009 pandemic, the virus spread fast and was a global threat due to modern transportation routes. Many people were hospitalized with severe clinical symptoms. Although influenza A generally causes upper respiratory tract illnesses in humans, the 2009 pandemic caused lower respiratory tract infections, as well as gastrointestinal symptoms (i.e., nausea, vomiting, and diarrhea), especially in children and healthy young adults 
. Our findings of virus isolates in the feces of mice challenged with most of the avian H1N1 viruses and diarrhea in some animals were consistent with the findings from the stool samples of patients during the 2009 pandemic 
. Observation of diarrhea in our study and high viral titers in the rectal swabs of ferrets infected with avian H1N1 virus raise concern about the potential shedding of the viruses in the feces and possible viral replication in the intestines. We still do not know whether the virus replicates in the intestines and is shed via the fecal route, or if it replicates only in the upper and lower respiratory tract and is simply displaced into the gastrointestinal tract via mucus secretion. Molecular analyses and detailed histopathologic examination are necessary to definitively answer this question.
This study serves as an initiative for a broader assessment. Here we have presented only the biological assessments of avian H1N1 viruses; further confirmation using molecular approaches such as genotyping and mutation analyses are needed. The genetic constellations of these viruses, the evolutionary distances among them, and the molecular changes in the virus genome that are associated with pathogenesis (i.e., potential pathogenicity markers), are areas that require further study. The implementation of a risk-assessment and risk-management program for avian H1N1 viruses worldwide would better estimate what to expect from nature and thus allow influenza researchers to be prepared to preserve public health.
The frequent circulation of H1N1 influenza A viruses in the human population and the potential pandemic risk that these viruses pose emphasize the necessity for a risk-assessment and risk-management program for H1N1 viruses residing in the wild bird population. The first step toward developing such a program would be the selection of a suitable small animal model in which to screen a large number of viruses to determine their potential pathogenicity and to facilitate a rapid response when warranted. As can be seen from our results, some avian H1N1 viruses residing in the natural reservoir can adapt and make the transition smoothly from birds to mammals, even without using an intermediate species. In fact, our findings in ferrets raise concern about the avian H1N1 viruses in terms of public health. More efficient surveillance studies and an efficient risk-assessment program for avian H1N1 viruses in nature are needed to identify the virulence levels by screening as many virus isolates as possible. Here we examined only the pathogenicity of avian H1N1 viruses of North American lineages, but pathogenicity is only one aspect of risk assessment. Therefore, a broader surveillance program would be indispensable for more efficient risk-assessment and risk-management of these and other influenza viruses. The significance of H1N1 viruses in wild bird populations and the vast diversity in that gene pool should be highlighted, because those viruses have the potential to bring a new pandemic strain into existence, either via direct transmission or mixing with other viruses that originate in a different host.