Pathogenesis studies have been performed in mice and other animal models in order to elucidate the H1N1pdm virus's molecular markers of virulence [19], [20]. Consistent with previous reports, we observed that the H1N1pdm virus was virulent for Balb/c mice, however only at high concentrations (≥10⁷ TCID₅₀). In our study, Balb/c mice infected intranasally with 5.4x10⁵ TCID₅₀ of Ca/04 showed significant body weight loss (∼20%) during the first 7 days post-infection (dpi), but eventually recovered (Fig 1A, B). This is in stark contrast to the 1918 pandemic strains, which are lethal for mice at concentrations significantly lower than 10⁵ TCID₅₀ [21]. In contrast to the Balb/c study, we found that Ca/04 was lethal for DBA/J2 mice, which succumbed to infection by 7 dpi at the same dose of 5.4×10⁵ TCID₅₀ (Fig 1A, B). This result is not necessarily unexpected since previous studies have shown that DBA/J2 mice have an increased susceptibility to influenza virus infection [17], [18], [22]. In order to determine whether the virus population lethal to DBA/J2 mice had an altered phenotype compared to the Ca/04 virus used as inoculum, lung homogenates from the affected DBA/J2 mice were used to infect three naïve Balb/c mice (DBA-Balb/c, Fig 1A, B). Interestingly, the virus contained in the DBA-mouse lung homogenates was lethal for Balb/c mice (Fig 1A, B). Balb/c mice succumbed to infection by 7 dpi, a timeframe that resembled the Ca/04 infection of DBA mice. The virus titer of the DBA-lung homogenates, used as inoculum for infection of Balb/c mice, was 1.2×10⁵ TCID₅₀, indicating that lethality in Balb/c mice was not due to an increased virus dose.

Because we could not rule out the possibility that the lethality of the virus present in the DBA-mouse lung homogenates was not due to the presence of cytokines and other substances toxic to Balb/c mice, lung homogenates from the Balb/c mice were grown in MDCK cells and the virus obtained was arbitrarily designated as mouse-adapted Ca/04 (ma-Ca/04). In vitro growth kinetics revealed that the ma-Ca/04 virus grew faster and yielded more than 10-fold higher titers than Ca/04 in MDCK cells (Fig 1C). To further characterize the ma-Ca/04 virus, pathogenesis and replication were evaluated in the Balb/c mouse and ferret models. The mouse 50% lethal dose (MLD₅₀) of ma-Ca/04 in Balb/c mice was 3.3 (log₁₀TCID₅₀), whereas that of the wild type virus was >6.0 (log₁₀TCID₅₀) indicating that indeed the virus had become more virulent for Balb/c mice (Table 1). Since we use virus from MDCK tissue culture supernatants, it is unlikely that cytokines or other substances are contributing to the ma-Ca/04 more lethal phenotype. The ma-Ca/04 also grew to higher titers in the lungs of mice compared to Ca/04 by 3 dpi (Fig 1D). H&E staining showed that the Ca/04 caused mild and limited alveolitis (Fig 1F). In contrast, lungs of mice infected with the ma-Ca/04 showed prominent lung pathology with severe alveolitis, characterized by the infiltration of neutrophils, lymphocytes and macrophages (Fig 1G). Immunohistochemistry for viral antigen showed more extensive positive staining in the bronchiolar lumen of ma-Ca/04-infected lungs than in the Ca/04-infected lungs (Figure S1 A, B, D and E). We also observed focal antigen staining in the alveolar area of the ma-Ca/04-infected lungs (Figure S1 F), whereas virus antigen was hardly detected in the alveolar area of Ca/04-infected lungs (Figure S1 C). In summary, these studies suggest that the ma-Ca/04 has increased virulence in the Balb/c mouse model when compared to Ca/04.

To determine whether mouse adaptation of the Ca/04 virus resulted in an altered fitness for other mammalian species, we compared the replication and transmission of ma-Ca/04 to Ca/04 in ferrets. Infected, direct contact and respiratory contact ferrets in the ma-Ca/04-infected and Ca/04-infected groups shed similar amounts of virus over time (Fig 2A, B). Peak virus titers were slightly higher in the ma-Ca/04 group however the differences were not statistically significant (not shown). Likewise, the transmission kinetics: onset of sneezing, peaks in body temperature, and body weight losses were similar between the two groups (Table S1 and Figure S2) and to our previous studies with Ca/04 [23]. The exception was one ferret inoculated with the ma-Ca/04 showed significant diarrhea and body weight loss and was humanely sacrificed at 6 dpi (Table S1). Replication and virus distribution in different tissues demonstrated that titers in the nasal turbinates of ferrets infected with ma-Ca/04 increased 10 fold over the Ca/04 group (Fig 2C). Furthermore, the virus was isolated from the olfactory bulb in one out of two ma-Ca/04 infected ferrets, whereas no virus was detected in the olfactory bulb from Ca/04-infected ferrets (Fig 2C). There was no obvious difference in titers from lung and trachea, and virus could not be isolated from the brain in either group (Fig 2C). These observations suggest that the ma-Ca/04 maintains its infectivity and transmissibility for ferrets at levels comparable to those observed with the Ca/04 virus.

To identify the molecular markers responsible for the virulence of ma-Ca/04 in Balb/c mice, the viral genome was sequenced and compared to Ca/04, as well as other H1N1pdm influenza strains. Sequence analysis revealed that only five amino acid substitutions had occurred during the adaptation of Ca/04: 3 in HA (D131E, S186P and A198E, based on the mature 1918 virus HA protein sequence [24], [25]), 1 in PA (E298K) and 1 in NP (D101G) (Fig 3 and Table 2). There were no amino acid mutations found in the PB2, PB1, NA, M and NS genes. The small number of mutations observed may be related to the low passage in mouse lungs as suggested previously [26], [27]. The D131E mutation sits outside the major antigenic site Sa and the receptor-binding site (RBS), potentially modulating access to the sialic acid receptor (Fig 3A, B, and C). The S186P mutation occurs within the RBS and so this mutation would have more of a role modulating access to the sialic acid than position 131. The S186P mutation overlaps also with antigenic site Sb (Fig 3A and C). Likewise, the A198E mutation sits within the edge of Sb, and is under immunological pressure (Fig 3B and C) [28], [29]. In fact, E198 is common in seasonal H1N1 strains [28], [29]. Further comparative sequence analysis showed that some current pandemic strains share the same D131E or S186P mutation in HA. As listed in Table 2, the amino acid at position 131 is E in strains A/Hiroshima/220/2009(H1N1) and A/Singapore/TLL52/2009(H1N1), and a mixture of D and E in strains A/New York/09/2009 (H1N1), A/New York/12/2009 (H1N1), A/New York/39/2009 (H1N1) and A/Ohio/07/2009 (H1N1). And the amino acid at position 186 is P in strains A/Ankara/17/2009 (H1N1), A/California/VRDL7/2009 (H1N1), A/Ontario/25913/2009 (H1N1) and A/Swine/NC/19646/2010 (H1N1), and a mixture of S and P in strains A/Kansas/03/2009 (H1N1) and A/Ontario/10016/2009 (H1N1). Likewise, the D101G mutation in the NP gene is found in the A/Mexico/4108/2009 (H1N1) strain, and the E298K mutation in the PA gene is found in the strain A/Korea/01/2009 (H1N1). These comparisons suggest that our adaptation scheme did not select for mutations unique to virus adaptation in mice. Instead, it implies that the Ca/04 may contain a mixed virus population, which was selected out in DBA/J2 mice. It is worth noting that two mutations in HA (D131E and S186P) are also highly conserved in the 1918 pandemic H1N1 strains sequenced to date (Table 2). It is tempting to speculate that amino acids at positions 131 and 186 are potential virulent determinants in H1N1 influenza viruses.

Although the ma-Ca/04 point mutations in ma-PA (E298K) and ma-NP (D101G) are present in naturally occurring H1N1pdm strains, they are not reflected in the consensus sequences for these two viral proteins. Therefore, we wanted to evaluate if such mutations affect polymerase activity. We assessed activity, using an influenza minigenome assay consisting of a secreted luciferase influenza replicon and plasmids encoding the polymerase complex and NP - as described in materials and methods. Polymerase activity was tested at different times post-transfection. The ma-PA and ma-NP were tested individually, as well as in combination, in the Ca/04 background, and compared to Ca/04 (Fig 4A). The ma-PA E298K mutation increased polymerase activity 2.0-fold compared to the wt PA from 24 h to 72 hours post-transfection (hpt), whereas the ma-NP D101G mutation (when tested without the ma-PA) reduced polymerase activity by ∼0.9 fold compared to the wt NP. The combined effect of ma-PA K298 and ma-NP G101 resulted in fold increases of ∼1.6 at 24 hpt, and ∼1.8, at 72 hpt over the wt, consistent with a ∼2 log₁₀ increase in growth kinetics for viruses carrying the ma-PA or the ma-PA and ma-NP mutations (in the context of the ma-Ca/04 HA gene) compared to the Ca/04 virus (Fig 4B).

To determine the role of each ma-Ca/04 mutation in the virulence phenotype, the HA, PA and NP genes from the ma-Ca/04 were cloned in a reverse genetics vector as previously described [30]. Four recombinant viruses (Table 1) were generated using the Ca/04 backbone. Pathogenesis studies in Balb/c mice indicated that the three mutations in HA caused significant morbidity and weight loss (maHA1:7Ca/04) however, the double mutants maHA-PA2:6Ca/04 and maHA-NP2:6Ca/04 were more virulent than maHA1:7Ca/04. The triple mutant, maHA-PA-NP3:5Ca/04, was the most virulent recombinant tested (Fig 4C), indicating all five mutations appear necessary to match the phenotype observed with ma-Ca/04. Interestingly, MLD₅₀ studies demonstrated that mutations in HA alone (maHA1:7Ca/04) contributed to a moderate increase in lethality; however, lethality increased in the context of the ma-PA and ma-NP. These results indicate a synergistic effect for the mutations in HA, PA and NP in ma-Ca/04 for virulence (Table 1).

In order to confirm whether the virulent phenotype imparted by mutations in the HA of ma-Ca/04, could modulate the virulence of related H1N1pdm viruses, we generated additional viruses in the backbone of A/Netherlands/602/09 (H1N1) –NL/602- (Table 1 and Fig 4D). We observed that the MLD₅₀ of wt NL/602 went from 6.0 to 3.4 when provided the maHA gene; maHA1:7NL/602 provided a MLD₅₀ similar to the ma-Ca/04 virus. Previous reports have shown that the NL/602 is lethal to Balb/c mice without prior adaptation (Fouchier R., pers communication). Our results are consistent with this observation, suggesting that the NL/602 virus contains a gene constellation more virulent than Ca/04. There are 8 amino acid differences between Ca/04 and NL/602: 3 in HA (P87S, T200A and I324V), 1 in PA (M581L), and 1 in NP (P224S). The T200A mutation is close to the A198E mutation found in ma-Ca/04 and just outside of antigenic site B [29]. The I324V lies outside the HA cleavage site, but its effects on cleavage remains to be determined. It also unknown whether the amino acid differences in PA and NP are responsible for the increased virulence of NL/602 in mice compared to the Ca/04. However, the virulence of NL/602 for mice can be greatly enhanced by incorporating the HA gene from the ma-Ca/04 virus. The mice inoculated with 8.0×10⁴TCID₅₀ of maHA1:7NL/602 succumbed to the infection by 5 dpi (Fig. 4D). To further determine the effect of the HA mutations in virulence, we created a recombinant virus carrying the HA gene from A/New York/18/2009 (H1N1) (NY/18) in the backbone of NL/602 (NY/18HA:7NL/602). The NY/18 virus contains D131, S186 and A198 in the HA, the same amino acids found in Ca/04 at those positions. The Ca/04 and NY/18 strains differ at HA positions (P87S, T200A, S206T and I324V). In this regard, the NL602 and NY/18 differ only at position S206T. As expected the MLD₅₀ of NY/18HA1:7NL/602 is >6.0 (Table 1). Mice infected with 8.0×10⁴TCID₅₀ of either NL/602 or NY/18HA1:7NL/602 lost roughly 20% and 15%, respectively, of their body weight within the first 7 dpi but eventually recovered (Fig 4D). These studies are consistent with the notion that mutations in the HA of mouse-adapted viruses can modulate virulence [26], [27].

Since the D131E and S186P mutations in the HA of ma-Ca/04 resemble amino acids present on the HA of the 1918 pandemic viruses (Table 2), we hypothesize that these mutations were responsible for the increased virulence of ma-Ca/04. To pinpoint the contribution of each amino acid mutation in HA to the virulence of ma-Ca/04, single and double mutants were generated using the remaining 7 genes from Ca/04 (1:7 recombinants). Balb/c mice (n = 5) infected with 8.0×10⁴TCID₅₀ of single mutant 186P, 131E, and 198E viruses showed average maximum bodyweight losses of 19%, 11% and 6%, respectively. In contrast, mice infected with 8.0×10⁴TCID₅₀ of Ca/04 lost only 3.9% of body weight (Fig 4E). These results demonstrate major contributions to virulence for amino acids at positions 131 and 186 in HA. Lung virus titers of mice inoculated with single mutant 186P, 131E, and 198E viruses were similar to those for Ca/04 at 3 dpi (Fig 4G), and were approximately 0.4_, 0.5, and 0.4 log₁₀ TCID₅₀ higher, respectively, than for Ca/04 at 6 dpi (Fig 4G). Lung virus titers were similar for the three single point mutants; however 186P and 131E viruses caused more morbidity than 198E virus indicating that virus replication alone cannot account for differences in virulence.

Mice infected with either double (131E/186P) or triple (131E/186P/198E) HA mutant virus lost weight rapidly. The average maximum body weight loss of mice infected with 131E/186P or 131E/186P/198E was 18% and 22%, respectively (Fig 4F). However, mice infected with mutants 131E/198E or 186P/198E lost 8% and 9% of their body weight, respectively (Fig 4F). These results suggest that the 131E/186P combination is the major contributor to virulence imparted by HA with the 198E mutation adding virulence only when present with the 131E/186P mutations. Lung virus titers for 131E/186P, 131E/198E and 131E/186P/198E were approximately 0.7, 0.6, and 0.6 log₁₀ TCID₅₀, respectively, higher than for Ca/04 at 3 dpi (Fig 4G). Additionally, lung virus titers for the 131E/186P, 131E/198E, 186P/198E, and 131E/186P/198E mutants were approximately 1.1, 0.9, 0.7 and 1.2 log₁₀ TCID₅₀ higher than for Ca/04 at 6 dpi (Fig 4G). Thus, these single and double mutants clearly demonstrate that the D131E and S186P mutations, alone and in combination, can increase the virulence of the pandemic H1N1 virus in Balb/c mice.

Given that positions 131, 186 and 198 in HA are located within or in close proximity to major antigenic sites and that 186 is within the RBS pocket (Fig 3), it was anticipated that these mutations could modulate antigenicity and receptor binding. Therefore, we used four ferret sera and two monoclonal antibodies (mAbs) raised against the H1N1pdm virus to evaluate the antigenic profile of ma-Ca/04 and compared it to other H1N1pdm viruses (Table 3 and data not shown). The four ferret sera showed a distinct HI profile dependent on the HA mutation. These sera had the lowest HI titer when HA protein carried 131E and/or 186P mutations (Table 3). The 131E/186P double mutation resulted in 7.4-fold lower HI titer than those obtained with the Ca/04 virus generated by reverse genetics. The 131E and 186P single mutants also displayed 4.5-fold and 3.9-fold lower HI titers, respectively, compared to the Ca/04 virus. In contrast, the single 198E mutation resulted in the highest HI titer (1.8-fold higher than with Ca/04). The HI profiles from 131E, 186P, 198E and 131E/186P mutant viruses were significantly different (p<0.01) than the one obtained for Ca/04. However, the HI titers from the double mutants, 131E/198E, 186P/198E, and the triple mutant, 131E/186P/198E, showed no statistical differences with the HI titer obtained for Ca/04 (p>0.05). Interestingly, one of the two monoclonal antibodies, mAb 5H7, had a different HI profile dependent on the HA mutations, mAb 3B2, showed no differences in the HA profile, regardless of the mutant used. The mAb 5H7 had the highest HI titers when the HA protein carried the 186P mutation, whether alone as the single mutant or in concert with the 131E and/or 131E/198E viruses. These viruses resulted in 8-fold higher titers than those obtained with Ca/04. The 131E single mutant also showed a 4-fold higher HI profile than the Ca/04. In contrast, the 198E single mutant had an HI titer 4-fold lower than the Ca/04 virus. It is also worth noting that the 198E mutation appears to affect HI titers using either the ferret polyclonal sera or the mAb 5H7 in the context of double mutations, either in association with 131E or 198P. Coincidentally, the HI profile analysis for these HA mutants follows the pattern of virulence of ma-Ca/04. Mutations at positions 131 and 186 had the highest impact by altering the antigenic make up of the molecule and its virulence.

We have shown that the D131E and S186P mutations increased the virulence for mice and conferred minor antigenic changes to the Ca/04 H1N1pdm virus. To determine the prevalence of such mutations we evaluated the host adaptation and/or host selection profile of H1 HA proteins from avian, swine, and human influenza viruses. From 1930 to 1998, 100% of H1 HAs from North American swine H1N1 isolates carried 131E, whereas 97% of them had 186P (including the prototype strain A/swine/Iowa/15/1930, SW30). North American swine H1N1 viruses started to carry the 131D and 186S mutations from 1999 onward. Analyses of swine H1 influenza sequences in Genbank indicate that from 1999 to 2009, North American swine H1N1 contain 81% 131D and 67% 186S. This data indicates a dominant presence of 131D and 186S in the recent North American swine H1N1 viruses, which have resulted through evolution in the swine population. In contrast, 100% of avian H1N1 influenza viruses, isolates collected from 1976 to 2009, carry 131E and 98% encode for 186P. Only one isolate, A/quail/Nanchang/12-340/2000 (H1N1), possesses 186S. Interestingly, up until 1919, 100% of human H1N1 strains contained 131E and 186P (1918-like H1N1 strains). Unfortunately there is a hiatus of influenza sequence information from 1920 to 1932; however, from 1933 to 1997, only 6% of the human H1 isolates contained 186P. The S186P mutation was prominent from 1998 to 2000 (86%) and now dominates since 2000 (99% isolates). In human strains, position 131 has transitioned from E131 to N131, and then to practically 100% of T131. With the exception of prior swine influenza strains that sporadically crossed to humans, no human H1N1 strain carried D131 until the introduction of H1N1pdm viruses. Since the 131E and 186P mutations were present in the 1918 H1N1 viruses of avian origin, we refer to these as avian-type mutations whereas the 131D and 186S mutations are labeled as swine-like mutations. Our analysis suggests that in humans, H1 influenza viruses have shown predilection for the 131E, 186P avian-type combination, which could lead to viruses with altered virulence phenotypes.

A/California/04/09 (H1N1) (Ca/04) and A/New York/18/09 (H1N1) (NY/18) were kindly provided by the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia. These viruses were provided as passage 2 viruses in MDCK cells. Passage 3 viruses were generated and stocks prepared and maintained at −70°C until use. Alternatively, stocks were prepared using viruses rescued by reverse genetics as indicated below. The A/Netherlands/602/09 has been previously described and was obtained by reverse genetics using plasmids kindly provided by Ron Fouchier, Erasmus Medical Center, The Netherlands [38]. Viruses were titrated in MDCK (Madin-Darby canine kidney) cells to determine the TCID₅₀ by the Reed and Muench method [39]. MDCK cells were maintained in Modified Eagle's medium (MEM) (Sigma-Aldrich, St. Louis, MO) containing 5% fetal bovine serum (FBS) (Sigma-Aldrich). 293-T human embryonic kidney cells were cultured in Opti-MEM I (GIBCO, Grand Island, NY) containing 5% FBS.

Five-week-old female mice (Balb/c and DBA/J2) (Charles River Laboratories) were anaesthetized with isofluorane before intranasal inoculation with 50 µl virus suspension. In an initial evaluation, DBA/J2 and Balb/c mice (n = 4) were infected intranasally with the Ca/04 virus (5.4×10⁵ TCID₅₀). Body weight changes and survival were recorded daily. Mice presenting ≥25% body weight loss were humanely euthanized and counted as dead. For adaptation studies lungs were collected from DBA/J2 mice, which succumbed to infection with Ca/04 (n = 2). Lungs were homogenized in PBS with antibiotics. After centrifugation at 6,000 rpm for 10 min, 50 µl of supernatant from the homogenate was passaged to naïve Balb/c mice (n = 3). Lungs from these infected Balb/c mice were then homogenized and inoculated into MDCK cells to prepare a virus stock. The 50% mouse lethal dose (MLD₅₀) was calculated using different virus doses in Balb/c and DBA mice (n = 3/per virus dose). Body weight and survival were recorded daily until 14 dpi. To evaluate the replication tropism of selected H1N1 pdm viruses in mice, lungs were collected at 3 and 5 dpi and titrated in MDCK cells. For histopathology analysis, mouse lungs collected at 3 dpi were fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). In order to evaluate differences in virulence of Ca/04 compared to related ma-Ca/04-derived mutants, the virus doses were adjusted to 8.0×10⁴ TCID₅₀/mouse.

The experimental design to study transmission of influenza in ferrets has been previously described [40]. Briefly, two ferrets (1/cage) were lightly anesthetized with ketamine (20 mg/kg) and xylazine (1 mg/kg) via an intramuscular injection and were inoculated intranasally with 10⁶ TCID₅₀ of virus in PBS, 250 µl per nostril. At 24 hpi, two naïve ferrets (1/cage) were introduced in direct contact with the infected ferret. A second ferret (1/group) was introduced as a respiratory contact separated from the infected and direct contact by a wire mesh wall with no possibility of physical contact. To monitor virus shedding and transmission nasal washes were collected daily and titrated in MDCK cells. Virus replication and tissue distribution were evaluated in groups of two ferrets/virus inoculated with 10⁶ TCID₅₀ of either Ca/04 or ma-Ca/04, respectively. Ferrets were euthanized at 4 dpi. Brain, olfactory bulb, nasal turbinate, trachea, and lungs were collected, homogenized and titrated in MDCK cells as described [41].

Animal studies were conducted under ABSL-3 conditions approved by USDA and performed according to the protocol R-09-93 “Transmissibility of Influenza A Viruses” approved by the Institutional Animal Care and Use Committee of the University of Maryland.

The vRNA and cDNA were prepared as previously described [30]. The eight fragments were amplified and sequenced using a combination of universal [42] and custom made primers (available upon request). Sequencing was performed using the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) on a 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.

The 8 gene segments of Ca/04 (passage 2 in MDCK cell) and ma-Ca/04 (stock, passage1 in MDCK cell) were amplified by RT-PCR and cloned in the bidirectional reverse genetics plasmid pDP2002 [30]. Mutations of interest in the HA gene were introduced using the QuickChange II site-directed mutagenesis kit (Stratagene, Inc., La Jolla, CA) according to manufacturer's protocols. The presence of each mutation was confirmed by sequencing. The recombinant viruses were recovered by transfection in co-cultured 293T and MDCK cells using eight plasmids as previously described [43]. Recovered viruses were sequenced to ensure the absence of unwanted mutations, titrated in MDCK cells and stocks were prepared and frozen at −70°C until use.

A model viral RNA (vRNA), consisting of the Gaussia Luciferase (GLuc) open reading frame flanked by the non-coding regions of the influenza NS segment was used to assess polymerase activity in a minigenome reconstitution assay. Briefly, 293T cells were seeded in 6-well plates and transfected with 1 µg of the reporter plasmid along with 1 µg of each of expression plasmids encoding PB2, PB1, PA and NP using the TransIT-LT1 (Mirus, Madison, WI) reagent following the recommendations of the manufacturer. In addition, the pCMV/SEAP plasmid, which encodes a secreted alkaline phosphatase gene, was co-transfected into the cells to normalize the transfection efficiency. At the indicated time points, supernatant from transfected cells were harvested and assayed for both luciferase and secreted alkaline phosphatase activities using either the BioLux Gaussia Luciferase Assay Kit (NEB, Ipswich, MA) or the Phospha-Light Secreted Alkaline Phosphatase Reporter Gene Assay System (A&D, Foster City, CA) according to the manufacturers' recommendation. Relative polymerase activity was calculated as the ratio of luciferase versus SEAP luminescence for three independent experiments with duplicate samples.

Graphs were produced and statistical analysis performed using the Prism software package (GraphPad Software Inc., La Jolla, CA).