Genomic Polymorphism of the Pandemic A (H1N1) Influenza Viruses Correlates with Viral Replication, Virulence, and Pathogenicity In Vitro and In Vivo

The novel pandemic A (H1N1) virus was first identified in Mexico in April 2009 and quickly spread worldwide. Like all influenzas, the H1N1 strain-specific properties of replication, virulence, and pathogenicity are a result of the particular genomic sequence and concerted expression of multiple genes. Thus, specific mutations may support increased virulence and may be useful as biomarkers of potential threat to human health. We performed comparative genomic analysis of ten strains of the 2009 pandemic A (H1N1) influenza viruses to determine whether genotypes associated with clinical phenotypes, which ranged from mild to severe illness and up to lethal. Virus replication capacity was tested for each strain in vitro using cultured epithelial cells, while virulence and pathogenicity were investigated in vivo using the BALB/c mouse model. The results indicated that A/Sichuan/1/2009 strain had significantly higher replication ability and virulence than the other strains, and five unique non-synonymous mutations were identified in important gene-encoding sequences. These mutations led to amino acid substitutions in HA (L32I), PA (A343T), PB1 (K353R and T566A), and PB2 (T471M), and may be critical molecular determinants for replication, virulence, and pathogenicity. Our results suggested that the replication capacity in vitro and virulence in vivo of the 2009 pandemic A (H1N1) viruses were not associated with the clinical phenotypes. This study offers new insights into the transmission and evolution of the 2009 pandemic A (H1N1) virus.


Introduction
The first influenza pandemic of the 21 st century was declared with the emergence of a novel influenza A (H1N1) strain in Mexico and the United States in April 2009 [1]. Genetic analysis of this novel virus revealed that it is composed of six gene segments which were derived from the triple-reassortant swine lineage and two others from the Eurasian avian-like swine lineage [1]. Thus far, the A (H1N1) influenza has caused a relatively mild pandemic, with a clinical spectrum ranging from slight upper respiratory tract irritation to severe pneumonia leading to acute respiratory distress syndrome [2]. Sporadic cases have occurred in which infection led to death, but those individuals most often had impaired immune status prior to influenza exposure. It is interesting to note that the Spanish influenza pandemic of 1918 and the Hong Kong influenza pandemic of 1968 were both characterized by a first wave of cases which elicited relatively mild illness, followed by a second wave of cases of fulminant disease [3]. The viral molecular mechanisms underlying this robust increase in disease severity have remained elusive; however, it has been hypothesized that pandemic viruses rely on advantageous genetic mutations to adapt to the human host upon zoonotic transmission. As a result, the newly evolved virus will generate a wave of more virulent cases than the first wave. Such genetic adaptation could also occur via gene reassortment events between co-circulating influenza A viruses in the human population.
The virulence, pathogenicity, and host range of influenza viruses have been intensely studied and many diverse factors have been implicated in each. In particular, virus-specific determinants encoded by the viral genome have been defined as principal components of virus survival and pathogenesis; these include the external surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), which interact with host membrane-bound sialic acids [4,5,6,7]. In addition, influenza encodes three polymerase proteins, which have been characterized as important determinants of strains H5N1 and H7N7and necessary for transmission of the 1918 H1N1 virus [5,6,8,9]. The two nonstructural proteins PB1-F2 [10,11] and NS1 [12] have also been implicated in the virulence capacities of H5N1 and 1918 H1N1 viruses. Intriguingly, genome sequence analysis of the 2009 pandemic A (H1N1) viruses revealed a striking absence of markers associated with high pathogenicity in avian and mammalian species, including the multibasic HA cleavage site [13] and the lysine at position 627 in the PB2 protein [14,15,16,17].
To better understand the potential consequences of viral genetic variations on infection characteristics, we investigated the genomic polymorphisms that occurred among ten strains of the 2009 pandemic A (H1N1) viruses. Virus replication was analyzed in an in vitro cell culture system, and virulence and pathogenicity were tested in vivo in a mouse model. The findings from our study provide insights into the functional contributions of viral genomic polymorphisms in virus replication, virulence, and pathogenicity, and implicate molecular evolution as a significant driving force behind the 2009 pandemic A (H1N1) influenza virus.

Viruses
The main background information for all ten virus strains is listed in Table

Mice challenge
Female 5-week-old specific pathogen-free BALB/c mice used in this study were obtained from the Institute of Laboratory Animal Sciences, Beijing, China. Mice were anesthetized and inoculated intranasally with virus (n = 23 per group; 50 ml of 10 6 TCID 50 ). In each group 10 mice were chosen at random for daily monitoring for signs of disease and mortality, up to 14 days post inoculation (d.p.i.). Ten of the remaining mice in each group were euthanized at 5 d.p.i. to obtain lung tissue biopsies for use in subsequent quantification of viral nucleotide material and pathological investigations. The final 3 mice were sacrificed at 5 d.p.i. and lungs were collected for viral titer detection. All procedures were approved by the Institute of Animal Use and Care Committee of the Institute of Laboratory Animal Science, Peking Union Medical College (approval number ILAS-PC-2010-002).

Plaque assay
The infectivity of viruses released from infected MDCK cells into supernatants and in the homogenized lung tissues of challenged mice were determined by plaque assay and expressed as log 10 plaque-forming units (PFU) per milliliter [20]. Briefly, confluent MDCK cells were incubated at 37uC for 1 hour with 10fold serial dilutions of virus. The cells were then washed and overlaid with minimum essential medium containing 0.3% bovine serum albumin (BSA), 0.9% Bacto agar, and 1 mg/ml TPCKtreated trypsin and antibiotics. Plaques were visualized by neutral red staining and counted after 48 hours of incubation at 37uC.

Real-time PCR
Total RNA was isolated from homogenized lung tissues by using the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. RNA was resuspended in 30 ml nuclease-free water and stored at 280uC. First-strand cDNA was synthesized from RNA (8 ml) by random primers in a SuperScript II reverse transcriptase (200 U) reaction mixture (20 ml) (Invitrogen). SYBR Green real-time quantitative PCR was performed with a StepOne PCR system (ABI) using cDNA (2 ml) and a reaction mixture (20 ml) containing 26 SYBR Green PCR Master Mix (10 ml) (ABI), 10 mM forward and reverse primers (1 ml each: SW-HA F786, 59-AATAACATTAGAAGCAACTGG-39; SW-HA R920, 59-AGGCTGGTGTTTATRGCACC-39), and nuclease-free water. The following thermal cycling conditions were used: 94uC for 3 min, followed by 35 cycles of 94uC for 30 s, 58uC for 30 s, and 72uC for 30 s.

Pathological analysis
Immediately following euthanasia, mouse lungs were removed, inflated, and fixed with 10% neutral buffered formalin overnight at 4uC. Subsequently, the formalin-preserved lung samples were embedded in paraffin and sectioned. Serial 4-mm sections were stained with Hematoxylin and Eosin (H&E) and examined for pathological changes that corresponded to infection. Images were obtained on an Olympus BX-50 light microscope at 406 magnification.

Receptor-binding assay
SA-a-2,6-N-acetyl lactosamine(69SLN-PAA)-Biotin, 69SLN-DI-PAA-Biotin were provided by The Scripps Research Institute (La Jolla, CA, USA). The receptor binding procedure was carried out as described elsewhere, with some modifications [21]. Briefly, 96-well flat-bottom polystyrene plates were coated with serial dilutions of sialyglycopolymers, and 32 HAU of live virus were added per well. Rabbit antisera against the A/California/ 07/2009 strain were diluted in phosphate buffered saline containing 1% bovine serum albumin and then added to each well. Bound antibody was detected by sequential additions of HRP-conjugated anti-rabbit IgG antibody and tetramethylbenzidine substrate solution and reading the spectrophotometric absorbance at 450 nm. Each sample was determined in triplicate.

DNA sequencing and analysis
All gene segments for each of the ten viruses were amplified by high fidelity PCR (KOD-Plus DNA polymerase; Toyobo, Japan). The PCR products were purified and sequenced (Invitrogen). Sequences of each gene of A/Guangdong/SWL28/2009 and A/ Sichuan-Wenjiang/SWL456/2009 viruses were deposited in GenBank. Sequences of all viruses were analyzed and aligned using ClustalW software (version 1.83).

Statistical analysis
Statistical analysis of the viral load and titer were performed using SPSS 11.5 software. The Duncan and least significant differences methods were used for comparisons among multiple groups by one-way ANOVA. The Kaplan-Meier method was used to estimate the probability of survival of infected mice. A probability value of 0.05 was considered as statistically significant.

In vitro comparison of virus replication in cells
Analysis of virus replication kinetics in MDCK cells revealed that all ten viruses reached their peak titers at 36 to 48 h.p.i, and the titers of A/Sichuan/1/2009 were significantly higher (P,0.05) than those obtained with other strains after 12 h.p.i (Fig. 1).

In vivo comparison of virulence in the mice model
To compare the virulence of the ten pandemic A (H1N1) influenza viruses in vivo, we intranasally challenged BALB/c mice with each virus and evaluated mortality, mean survival days, total body weight loss, viral RNA loads and titers in lung tissues. The percentages of mice that survived the infections are shown in Figure 2A.  (Fig. 2C).
To further investigate the differences in pathogenicity of each virus, we determined the degree of virus replication occurring in lung tissues of challenged mice. The mean viral RNA loads (copy numbers per mg 6 standard deviation (SD); Fig. 2D) and viral titers (log 10 PFU per mg; Fig. 2E) were determined at day 5 postinfection, when virus shedding is known to reach its peak according to our previous studies [23,24]. Mice inoculated with the A/Sichuan/1/2009 virus presented with the highest RNA copy numbers of virus in the lungs and significantly higher (P,0.05) viral RNA load than the other nine viruses. Consistent with the viral RNA load result, lung tissues of mice challenged with A/Sichuan/1/2009 exhibited the highest viral titer.
In order to examine the differential pathological changes that may take place in the lungs of mice challenged with the different strains of virus, lung tissues were isolated day 5 post-infection. All viruses were found to have replicated efficiently in the lung tissues. In addition, all lung tissue samples exhibited characteristic pathology of influenza infection, including inflammatory hyperaemia, hemorrhage, edema, and exudative pathological changes.

Sequence alignment of A (H1N1) viral genomes
The full-length sequences of all ten viral genomes were obtained by high fidelity PCR and sequenced as described in the Methods section. The sequences of the HA, NA, M2&M1, NEP&NS1, NP, PA, PB1, and PB2 genes in A/Guangdong/SWL28/2009 and A/ Sichuan-Wenjiang/SWL456/2009 viruses were deposited in GenBank under accession numbers HM051340 to HM051347, and HM051348 to HM051355, respectively. Genomic polymorphisms found in each of the ten viruses are listed in Table 2. In particular, unique non-synonymous mutations were found in some of the virulence genes of A/Sichuan/1/2009: a Leu-to-Ile variation at position 32 in the HA coding sequence and another four substitutions in the polymerases: Ala-to-Thr at position 343 in PA, Lys-to-Arg at position 353 and Thr-to-Ala at position 566 in PB1, and Thr-to-Met at position 471 in PB2.

Effect of HA Leu32Ile substitution on receptor binding ability
Previous studies have suggested that HA mutations may act to increase virus virulence by affecting the affinity of HA binding to host sialyl receptors. We examined three representative strains (A/ California/04/2009, A/Sichuan/1/2009, A/Beijing/3/2009) and measured their HA receptor binding abilities to synthetic sialic substrates. No significant difference was observed in the affinities of the three HAs for binding to human-type 69SLN or 69DI-SLN and avian-type 39SL or 39DI-SL receptors (P.0.05) (Fig. 4). This finding demonstrated that the HA Leu32Ile mutation in A/ Sichuan/1/2009 contributed minimally, or not at all, to the uniquely high virulence phenotype.

Discussion
Influenza viruses cause epidemics and pandemics through antigenic drift or antigenic shift [25]. The 2009 pandemic A (H1N1) virus has generally been associated with mild disease and a relatively low mortality rate; however, sporadic severe or fatal cases have been reported. Certainly, discrepancies in individual immunity may support such diverse virulence of the 2009 pandemic A (H1N1) viruses, but it is as likely that some yet undefined viral molecular mechanisms are at play [3,26]. Mutations in specific regions of a given viral genome are known to result in increased virulence, and may lead to a more severe pandemic. In this study, we investigated the molecular basis of the 2009 pandemic A (H1N1) virus that mediates its uniquely robust characteristics of replication, virulence, and pathogenicity.
We first examined the viral replication kinetics of all ten viruses by using an in vitro infection model of MDCK cells. The A/ Sichuan/1/2009 strain had an obvious enhanced replication ability, which led to increased peak titers of roughly 3-4 orders of magnitude higher than the other viruses, with the exception of A/ California/04/2009 and A/California/07/2009 strains. These two viruses exhibited 2-3 orders of magnitude lower viral titers, as compared with A/Sichuan/1/2009 after 12 h.p.i. We also performed in vivo analysis using the BALB/c mouse model. Pronounced virulence and pathogenicity of the A/Sichuan/1/ 2009 virus were both observed; mice infected with this virus had the lowest survival ratio, shortest mean survival days, most extensive body weight loss, highest virus RNA copy numbers and titers in lung tissues, most severe pathological changes, and largest lesion areas in lung tissues. Taken together, these in vitro and in vivo results indicated that the A/Sichuan/1/2009 virus has the strongest replication ability in MDCK cells and virulence in the BALB/c mouse model. Sequence alignment among the ten A (H1N1) viruses revealed that the A/Sichuan/1/2009 virus genome harbors five unique mutations: L32I in HA; A343T in PA; K353R and T566A in PB1; and T471M in PB2. Receptor binding assay showed that despite having a non-synonymous mutation in the HA gene, the A/ Sichuan/1/2009 virus did not have increased binding affinity for synthetic sialyl receptors, including the human type 69SLN and the avian type 39SL. Thus, it is unlikely that the HA Leu32Ile mutation acts as a key molecular determinant for the increased virulence of A/Sichuan/1/2009. While previous studies have associated another mutation in the HA receptor binding domain, Asp222Gly, with enhanced virulence [24,27,28,29,30,31], it remains to be confirmed whether HA affinity for sialyl receptors is directly responsible for the enhanced virulence of the 2009 pandemic A (H1N1) influenza viruses. However, Glinsky and Melidou et al. presented findings to suggest that the HA Gln293His amino acid change may be associated with increased disease severity [32,33].
The polymerase genes of influenza viruses are considered to be extremely important for virulence. Codon position 515 in the PA protein, for example, is significantly related to pathogenicity of an H5N1 virus in ducks [34]. A reverse genetics study demonstrated that mutations in the PB2 protein, Glu627Lys and Asp701Asn, were responsible for virulence in mammalian species [8]. The amino acid at position 701 in PB2 has been characterized as crucial to replication and lethality of duck-originated H5N1 viruses in mice [35]. The same PB2 amino acid residue was shown to contribute to increased lethality of an H7N1 avian influenza virus in a mouse model [36]. However, Jagger et al. reported that influenza viruses containing mutations at residues 627 and 701 in the context of the pandemic A (H1N1) virus polymerase complex have attenuated virulence both in cell culture and the mouse  model system [17]. Recently, the PB2 mutation Thr271Ala was found to enhance polymerase activity and viral growth in human cells [37]. PB2 residue 158 has been described as a pathogenic determinant of the pandemic H1N1 and H5 influenza A viruses in mice [38]. Moreover, PB1-F2, a short viral protein of approximately 90 amino acids expressed from a +1 reading frame in the PB1 gene segment, was also reported as another virulence determinant; the serine at position 66 in PB1-F2 was associated with increased disease pathology in a mouse model [11,39,40] [41,42]. All the above data suggest that the polymerase complex mediates virulence of influenza viruses. Indeed, the four residues mutated within the polymerase complex of A/Sichuan/1/2009 virus may indicate key virulence determinants of the 2009 pandemic A (H1N1) viruses. However, it is still uncertain whether the increased virulence is caused by any single substitution or some combinations of the four. Further investigations using reverse genetics are likely to provide insights into this question.
Epidemiologic analysis showed that the ten viruses examined in this study could be divided into three groups based on time and region of prevalence. Group I contained the A/California/04/ 2009 and A/California/07/2009 viruses, which represented the very first 2009 pandemic A (H1N1) influenza patients reported. Group II contained the A/Sichuan/1/2009 strain, which was isolated from the first reported case in China. As these Group II strains were isolated during the earliest phase of the Chinese pandemic, this group also represented the period of H1N1 global dissemination [43]. Group III contained the other seven strains, which were representative of domestic infections and were all collected in China. The same HA sequence polymorphism found in A/Sichuan/1/2009 existed in other 2009 pandemic A (H1N1) strains which were collected across the globe from April to June of 2009. Most of these other strains represented the first waves of H1N1 in their respective countries, including Canada, Mexico, Nicaragua, France, Finland, and the United States. Interestingly, PA mutation in A/Sichuan/1/2009 has never been reported before. In contrast, sequence polymorphisms in PB1 and PB2 have been identified in strains collected from the first waves in Canada, Nicaragua, and some cities of the United States, but little information is available about the virulence and pathogenicity of those particular strains.
Belser et al. also used a mouse model to evaluate the virulence of a collection of 2009 A (H1N1) viruses. Their studies ultimately demonstrated that the viruses exhibited mild to moderate virulence in mice. They also performed sequence analysis of their isolates, and identified similar polymorphisms to those reported here; molecular features which are frequently found among viruses of high pathogenicity in mammalian models were not detected in either their or our studies [44]. Although it was reported that hypercytokinemia is not a general feature of infection with the 2009 pandemic A (H1N1) viruses, including those isolated from fatal cases [44], however, it is likely that some, at least subtle, affects from the cytokine storm exist to the virulence [45,46,47,48,49,50]. Since none of the genetic changes identified in A/Sichuan/1/2009 involve critical domain/motifs in HA or in the polymerase complex [8,51,52,53], further study addressing the potential for host-based differences in immunologic response is warranted.
Our results also suggested that the virulence of the 2009 pandemic A (H1N1) viruses were not associated with the clinical phenotypes of the corresponding patients; A/Sichuan-Wenjiang/ SWL456/2009 and A/Guangdong/SWL28/2009, viruses collected from a deceased and severely ill patient respectively, showed much lower replication ability, virulence, and pathogenicity than A/Sichuan/1/2009, a virus from a patient with mild symptoms. Previous investigations into a series of severe and fatal cases of the pandemic H1N1 influenza revealed that pregnancy, obesity, diabetes, and other co-morbid conditions were associated with severe disease [54,55]. Therefore, it is reasonable to hypothesize that the two patients with A/Sichuan-Wenjiang/SWL456/2009 or A/Guangdong/SWL28/2009 virus infection were suffering from concomitant complicating diseases. Unfortunately, time and anonymity concerns have made it impossible to now trace the clinical features and anamnesis of these two patients. It is interesting to note, here, that Belser et al. deduced the same conclusion, citing that 2009 H1N1 viruses isolated from fatal cases did not demonstrate enhanced virulence in a mouse model as compared with isolates from mild human cases [44].
In summary, we found that the genomic polymorphisms that characterize each of the 2009 pandemic A (H1N1) influenza viruses contribute to virus replication ability, virulence, and pathogenicity. Non-synonymous polymorphisms associated with amino acid substitutions in PA (A343T), PB1 (K353R and T566A), and PB2 (T471M) were identified as potential key virulence determinants. Meanwhile, the replication ability in vitro and virulence in vivo of the 2009 pandemic A (H1N1) viruses were not associated with the clinical phenotypes exhibited by the corresponding patients. These preliminary observations contribute to our understanding of the genetic process of transmission and evolution of the 2009 pandemic A (H1N1) influenza viruses. Because of the functional importance of the polymerase complex in replication, virulence, and pathogenicity, the mutations described here deserve further investigation and may lead to discoveries of new vaccines and therapeutic drugs against this globally important virus.

Author Contributions
Conceived and designed the experiments: CQ LX. Performed the