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1.  Mutation Analysis of 2009 Pandemic Influenza A(H1N1) Viruses Collected in Japan during the Peak Phase of the Pandemic 
PLoS ONE  2011;6(4):e18956.
Pandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges.
A total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations.
Results and Conclusions
Our sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo.
PMCID: PMC3084724  PMID: 21572517
2.  Frequency of D222G haemagglutinin mutant of pandemic (H1N1) pdm09 influenza virus in Tunisia between 2009 and 2011 
Diagnostic Pathology  2013;8:124.
The novel pandemic A (H1N1) pdm09 virus was first identified in Mexico in April 2009 and since then it spread worldwide over a short period of time. Although the virus infection is generally associated with mild disease and a relatively low mortality, it is projected that mutations in specific regions of the viral genome, especially within the receptor binding domain of the haemagglutinin (HA) protein could result in more virulent virus stains, leading to a more severe pathogenicity.
To monitor the genetic polymorphisms at position 222 of Haemagglutinin of influenza A(H1N1)pdm09 viruses from both outpatients with mild influenza and individuals with severe disease requiring hospitalization, during 2009–2010 and 2010–2011 seasons, a sequence-based genotypic assessment of viral populations to understand the prevalence of D222G mutation.
The D222G was identified in clinical specimens from 3 out of 42 cases analyzed in Tunisia with severe outcome (7%). Interestingly, in one fatal case out of four viruses taken from fatal cases studied (25%). Also this mutation was found in one mild case out of 8 mild cases studied (0.1%). D222E substitution was found in virus taken from one patient with severe clinical syndrome (2%) out of 42 severe cases analyzed and E374K substitution was found in two severe cases (4%) out of 42 severe cases studied.
A specific mutation in the viral haemagglutinin (D222G) was found in fatal, severe and mild case. Further virological, clinical and epidemiological investigations are needed to ascertain the role of this and other mutations that may alter the virulence and transmissibility of the pandemic influenza A (H1N1)pdm09.
Virtual Slides
The virtual slide(s) for this article can be found here:
PMCID: PMC3751102  PMID: 23902660
D222G substitution; Haemagglutinin; Influenza A(H1N1)pdm09 virus; Pandemic; Severe respiratory infection
3.  Influenza A/H1N1 2009 Pandemic and Respiratory Virus Infections, Beijing, 2009–2010 
PLoS ONE  2012;7(9):e45807.
To determine the role of the pandemic influenza A/H1N1 2009 (A/H1N1 2009pdm) in acute respiratory tract infections (ARTIs) and its impact on the epidemic of seasonal influenza viruses and other common respiratory viruses, nasal and throat swabs taken from 7,776 patients with suspected viral ARTIs from 2006 through 2010 in Beijing, China were screened by real-time PCR for influenza virus typing and subtyping and by multiplex or single PCR tests for other common respiratory viruses. We observed a distinctive dual peak pattern of influenza epidemic during the A/H1N1 2009pdm in Beijing, China, which was formed by the A/H1N1 2009pdm, and a subsequent influenza B epidemic in year 2009/2010. Our analysis also shows a small peak formed by a seasonal H3N2 epidemic prior to the A/H1N1 2009pdm peak. Parallel detection of multiple respiratory viruses shows that the epidemic of common respiratory viruses, except human rhinovirus, was delayed during the pandemic of the A/H1N1 2009pdm. The H1N1 2009pdm mainly caused upper respiratory tract infections in the sampled patients; patients infected with H1N1 2009pdm had a higher percentage of cough than those infected with seasonal influenza or other respiratory viruses. Our findings indicate that A/H1N1 2009pdm and other respiratory viruses except human rhinovirus could interfere with each other during their transmission between human beings. Understanding the mechanisms and effects of such interference is needed for effective control of future influenza epidemics.
PMCID: PMC3447804  PMID: 23029253
4.  Amino Acids Transitioning of 2009 H1N1pdm in Taiwan from 2009 to 2011 
PLoS ONE  2012;7(9):e45946.
A swine-origin influenza A was detected in April 2009 and soon became the 2009 H1N1 pandemic strain (H1N1pdm). The current study revealed the genetic diversity of H1N1pdm, based on 77 and 70 isolates which we collected, respectively, during the 2009/2010 and 2010/2011 influenza seasons in Taiwan. We focused on tracking the amino acid transitioning of hemagglutinin (HA) and neuraminidase (NA) genes in the early diversification of the virus and compared them with H1N1pdm strains reported worldwide. We identified newly emerged mutation markers based on A/California/04/2009, described how these markers shifted from the first H1N1pdm season to the one that immediately followed, and discussed how these observations may relate to antigenicity, receptor-binding, and drug susceptibility. It was found that the amino acid mutation rates of H1N1pdm were elevated, from 9.29×10−3 substitutions per site in the first season to 1.46×10−2 in the second season in HA, and from 5.23×10−3 to 1.10×10−2 in NA. Many mutation markers were newly detected in the second season, including 11 in HA and 8 in NA, and some were found having statistical correlation to disease severity. There were five noticeable HA mutations made to antigenic sites. No significant titer changes, however, were detected based on hemagglutination inhibition tests. Only one isolate with H275Y mutation known to reduce susceptibility to NA inhibitors was detected. As limited Taiwanese H1N1pdm viruses were isolated after our sampling period, we gathered 8,876 HA and 6,017 NA H1N1pdm sequences up to April 2012 from NCBI to follow up the dynamics of mentioned HA mutations. While some mutations described in this study seemed to either settle in or die out in the 2011–2012 season, a number of them still showed signs of transitioning, prompting the importance of continuous monitoring of this virus for more seasons to come.
PMCID: PMC3454337  PMID: 23029335
5.  Development of Two Types of Rapid Diagnostic Test Kits To Detect the Hemagglutinin or Nucleoprotein of the Swine-Origin Pandemic Influenza A Virus H1N1▿  
Since its emergence in April 2009, pandemic influenza A virus H1N1 (H1N1 pdm), a new type of influenza A virus with a triple-reassortant genome, has spread throughout the world. Initial attempts to diagnose the infection in patients using immunochromatography (IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm but not seasonal influenza A (H1N1 and H3N2) or B viruses. Using two of these antibodies, one recognizing viral hemagglutinin (HA) and the other recognizing nucleoprotein (NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses. The specificities of both IC test kits were very high (93% for the HA kit, 100% for the NP kit). The test sensitivities for detection of H1N1 pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A virus detection assay. Use of the anti-NP antibody could allow the rapid and accurate diagnosis of H1N1 pdm infections.
PMCID: PMC3067372  PMID: 21228147
6.  Reconstruction of the Evolutionary Dynamics of the A(H1N1)pdm09 Influenza Virus in Italy during the Pandemic and Post-Pandemic Phases 
PLoS ONE  2012;7(11):e47517.
The aim of this study was to reconstruct the evolutionary dynamics of the A(H1N1)pdm09 influenza virus in Italy during two epidemic seasons (2009/2010 and 2010/2011) in the light of the forces driving the evolution of the virus. Nearly six thousands respiratory specimens were collected from patients with influenza-like illness within the framework of the Italian Influenza Surveillance Network, and the A(H1N1)pdm09 hemagglutinin (HA) gene was amplified and directly sequenced from 227 of these. Phylodynamic and phylogeographical analyses were made using a Bayesian Markov Chain Monte Carlo method, and codon-specific positive selection acting on the HA coding sequence was evaluated. The global and local phylogenetic analyses showed that all of the Italian sequences sampled in the post-pandemic (2010/2011) season grouped into at least four highly significant Italian clades, whereas those of the pandemic season (2009/2010) were interspersed with isolates from other countries at the tree root. The time of the most recent common ancestor of the strains circulating in the pandemic season in Italy was estimated to be between the spring and summer of 2009, whereas the Italian clades of the post-pandemic season originated in the spring of 2010 and showed radiation in the summer/autumn of the same year; this was confirmed by a Bayesian skyline plot showing the biphasic growth of the effective number of infections. The local phylogeography analysis showed that the first season of infection originated in Northern Italian localities with high density populations, whereas the second involved less densely populated localities, in line with a gravity-like model of geographical dispersion. Two HA sites, codons 97 and 222, were under positive selection. In conclusion, the A(H1N1)pdm09 virus was introduced into Italy in the spring of 2009 by means of multiple importations. This was followed by repeated founder effects in the post-pandemic period that originated specific Italian clades.
PMCID: PMC3494699  PMID: 23152755
7.  Surveillance of influenza viruses in the post-pandemic era (2010–2012) in Northern Italy 
The activity and circulation of influenza viruses in Lombardy — Northern Italy — (a region with nearly 10 out of the 60 million inhabitants of Italy) were investigated during two consecutive seasons (2010–2011 and 2011–2012), as part of the Italian Influenza Surveillance Network. The molecular characteristics of the hemagglutinin (HA) sequence of circulating viruses were analyzed to investigate the emergence of influenza viral variants. In the surveyed area, the influenza activity of these two post-pandemic seasons was similar in terms of both time frame and impact. The timing of the influenza epidemics was similar to the timing seen prior to the emergence of the pandemic A(H1N1) virus in 2009. A(H1N1)pdm09 was the predominant virus circulating during the 2010–2011 post-pandemic season and then—unexpectedly—almost disappeared. The HA sequences of these A(H1N1)pdm09 viruses segregated in a different genetic group with respect to those identified during the 2009 pandemic, although they were still closely related to the vaccine viral strain A/California/07/2009. Influenza A(H3N2) viruses were the predominant viruses circulating during the 2011–2012 season, accounting for nearly 88% of influenza viruses identified. All HA sequences of the A(H3N2) viruses isolated in the 2011–2012 season fell into the A/Victoria/208/2009 genetic clade (although the A/Perth/16/2009 virus was the reference vaccine strain). B viruses presented with a mixed circulation of viral variants during these two seasons: viruses belonging to both B/Victoria and B/Yamagata lineages co-circulated in different proportions, with a notable rise in the proportion of B/Yamagata viruses (B/Wisconsin/1/2010-like) during the 2011–2012 epidemic. In conclusion, the continuous monitoring of the characteristics of circulating viruses is an essential tool for understanding the epidemiological and virological features of influenza viruses, for monitoring their matching with seasonal vaccine strains, and for tuning vaccination strategies.
PMCID: PMC3891725  PMID: 23302775
Influenza virus; influenza surveillance network; epidemiology; variants; hemagglutinin; phylogeny; influenza vaccination
8.  Comparative Analyses of Pandemic H1N1 and Seasonal H1N1, H3N2, and Influenza B Infections Depict Distinct Clinical Pictures in Ferrets 
PLoS ONE  2011;6(11):e27512.
Influenza A and B infections are a worldwide health concern to both humans and animals. High genetic evolution rates of the influenza virus allow the constant emergence of new strains and cause illness variation. Since human influenza infections are often complicated by secondary factors such as age and underlying medical conditions, strain or subtype specific clinical features are difficult to assess. Here we infected ferrets with 13 currently circulating influenza strains (including strains of pandemic 2009 H1N1 [H1N1pdm] and seasonal A/H1N1, A/H3N2, and B viruses). The clinical parameters were measured daily for 14 days in stable environmental conditions to compare clinical characteristics. We found that H1N1pdm strains had a more severe physiological impact than all season strains where pandemic A/California/07/2009 was the most clinically pathogenic pandemic strain. The most serious illness among seasonal A/H1N1 and A/H3N2 groups was caused by A/Solomon Islands/03/2006 and A/Perth/16/2009, respectively. Among the 13 studied strains, B/Hubei-Wujiagang/158/2009 presented the mildest clinical symptoms. We have also discovered that disease severity (by clinical illness and histopathology) correlated with influenza specific antibody response but not viral replication in the upper respiratory tract. H1N1pdm induced the highest and most rapid antibody response followed by seasonal A/H3N2, seasonal A/H1N1 and seasonal influenza B (with B/Hubei-Wujiagang/158/2009 inducing the weakest response). Our study is the first to compare the clinical features of multiple circulating influenza strains in ferrets. These findings will help to characterize the clinical pictures of specific influenza strains as well as give insights into the development and administration of appropriate influenza therapeutics.
PMCID: PMC3217968  PMID: 22110664
9.  Molecular Characterization of the Predominant Influenza A(H1N1)pdm09 Virus in Mexico, December 2011–February 2012 
PLoS ONE  2012;7(11):e50116.
When the A(H1N1)pdm09 pandemic influenza virus moved into the post-pandemic period, there was a worldwide predominance of the seasonal influenza A(H3N2) and B viruses. However, A(H1N1)pdm09 became the prevailing subtype in the 2011–2012 influenza season in Mexico and most of Central America. During this season, we collected nasopharyngeal swabs of individuals presenting with influenza-like illness at our institution in Mexico City. Samples were tested for seasonal A(H3N2) and B influenza viruses, as well as A(H1N1)pdm09 by real-time reverse transcription–polymerase chain reaction. Of 205 samples tested, 46% were positive to influenza, all of them A(H1N1)pdm09. The clinical characteristics of patients showed a similar pattern to the 2009 pandemic cases. Using next generation sequencing, we obtained whole genome sequences of viruses from 4 different patients, and in 8 additional viruses we performed partial Sanger sequencing of the HA segment. Non-synonymous changes found in the Mexican isolates with respect to the prototype isolate H1N1 (A/California/04/2009) included HA S69T, K163R and N260D unique to 2012 Mexican and North American isolates and located within or adjacent to HA antigenic sites; HA S143G, S185T, A197T and S203T previously reported in viruses from the 2010–2011 season, located within or adjacent to HA antigenic sites; and HA E374K located in a relevant site for membrane fusion. All Mexican isolates had an oseltamivir-sensitive genotype. Phylogenetic analysis with all 8 influenza gene segments showed that 2012 Mexican sequences formed a robust, distinct cluster. In all cases, 2012 Mexican sequences tended to group with 2010–2011 Asian and European sequences, but not with 2009 Mexican sequences, suggesting a possible recent common ancestor between these latter regions and the 2012 Mexican viruses. It remains to be defined if these viral changes represent an important antigenic drift that would enable viral immune evasion and/or affect influenza vaccine effectiveness.
PMCID: PMC3510220  PMID: 23209653
10.  Epidemiological aspects of influenza A related to climatic conditions during and after a pandemic period in the city of Salvador, northeastern Brazil 
Memórias do Instituto Oswaldo Cruz  2014;109(2):229-235.
During the influenza pandemic of 2009, the A(H1N1)pdm09, A/H3N2 seasonal and influenza B viruses were observed to be co-circulating with other respiratory viruses. To observe the epidemiological pattern of the influenza virus between May 2009-August 2011, 467 nasopharyngeal aspirates were collected from children less than five years of age in the city of Salvador. In addition, data on weather conditions were obtained. Indirect immunofluorescence, real-time transcription reverse polymerase chain reaction (RT-PCR), and sequencing assays were performed for influenza virus detection. Of all 467 samples, 34 (7%) specimens were positive for influenza A and of these, viral characterisation identified Flu A/H3N2 in 25/34 (74%) and A(H1N1)pdm09 in 9/34 (26%). Influenza B accounted for a small proportion (0.8%) and the other respiratory viruses for 27.2% (127/467). No deaths were registered and no pattern of seasonality or expected climatic conditions could be established. These observations are important for predicting the evolution of epidemics and in implementing future anti-pandemic measures.
PMCID: PMC4015250  PMID: 24714967
influenza; A(H1N1)pdm09; children; epidemiology; seasonality; climate
11.  Development of an Immunochromatographic Assay Specifically Detecting Pandemic H1N1 (2009) Influenza Virus ▿  
Journal of Clinical Microbiology  2010;48(3):703-708.
The pandemic caused by a new type of influenza virus, pandemic H1N1 (2009) influenza virus A (AH1pdm), has had a major worldwide impact. Since hemagglutinin (HA) genes are among the most specific genes in the influenza virus genome, AH1pdm can be definitively diagnosed by viral gene analysis targeting the HA genes. This type of analysis, however, cannot be easily performed in clinical settings. While commercially available rapid diagnosis kits (RDKs) based on immunochromatography can be used to detect nucleoproteins (NPs) of influenza A and B viruses in clinical samples, there are no such kits that are specific for AH1pdm. We show here that an RDK using a combination of monoclonal antibodies against NP can be used to specifically detect AH1pdm. The RDK recognized AH1pdm virus isolates but did not recognize seasonal H1N1 and H3N2 and influenza B viruses, indicating that the specificity of the RDK is 100%. A parallel comparison of RDK with a commercial influenza A/B virus kit revealed that both types of kits had equal sensitivities in detecting their respective viruses. Preliminary evaluation of clinical samples from 5 individuals with PCR-confirmed human AH1pdm infection showed that the RDK was positive for all samples, with the same detection intensity as that of a commercial influenza A/B virus kit. This RDK, together with a new vaccine and the stockpiling of anti-influenza drugs, will make aggressive measures to contain AH1pdm infections possible.
PMCID: PMC2832444  PMID: 20071549
12.  Virological Surveillance of Influenza Viruses during the 2008–09, 2009–10 and 2010–11 Seasons in Tunisia 
PLoS ONE  2013;8(9):e74064.
The data contribute to a better understanding of the circulation of influenza viruses especially in North-Africa.
The objective of this surveillance was to detect severe influenza cases, identify their epidemiological and virological characteristics and assess their impact on the healthcare system.
We describe in this report the findings of laboratory-based surveillance of human cases of influenza virus and other respiratory viruses' infection during three seasons in Tunisia.
The 2008–09 winter influenza season is underway in Tunisia, with co-circulation of influenza A/H3N2 (56.25%), influenza A(H1N1) (32.5%), and a few sporadic influenza B viruses (11.25%). In 2010–11 season the circulating strains are predominantly the 2009 pandemic influenza A(H1N1)pdm09 (70%) and influenza B viruses (22%). And sporadic viruses were sub-typed as A/H3N2 and unsubtyped influenza A, 5% and 3%, respectively. Unlike other countries, highest prevalence of influenza B virus Yamagata-like lineage has been reported in Tunisia (76%) localised into the clade B/Bangladesh/3333/2007. In the pandemic year, influenza A(H1N1)pdm09 predominated over other influenza viruses (95%). Amino acid changes D222G and D222E were detected in the HA gene of A(H1N1)pdm09 virus in two severe cases, one fatal case and one mild case out of 50 influenza A(H1N1)pdm09 viruses studied. The most frequently reported respiratory virus other than influenza in three seasons was RSV (45.29%).
This article summarises the surveillance and epidemiology of influenza viruses and other respiratory viruses, showing how rapid improvements in influenza surveillance were feasible by connecting the existing structure in the health care system for patient records to electronic surveillance system for reporting ILI cases.
PMCID: PMC3777972  PMID: 24069267
13.  Influenza Epidemiology and Vaccine Effectiveness among Patients with Influenza-Like Illness, Viral Watch Sentinel Sites, South Africa, 2005–2009 
PLoS ONE  2014;9(4):e94681.
There is limited data on the epidemiology of influenza and few published estimates of influenza vaccine effectiveness (VE) from Africa. In April 2009, a new influenza virus strain infecting humans was identified and rapidly spread globally. We compared the characteristics of patients ill with influenza A(H1N1)pdm09 virus to those ill with seasonal influenza and estimated influenza vaccine effectiveness during five influenza seasons (2005–2009) in South Africa.
Epidemiological data and throat and/or nasal swabs were collected from patients with influenza-like illness (ILI) at sentinel sites. Samples were tested for seasonal influenza viruses using culture, haemagglutination inhibition tests and/or polymerase chain reaction (PCR) and for influenza A(H1N1)pdm09 by real-time PCR. For the vaccine effectiveness (VE) analysis we considered patients testing positive for influenza A and/or B as cases and those testing negative for influenza as controls. Age-adjusted VE was calculated as 1-odds ratio for influenza in vaccinated and non-vaccinated individuals.
From 2005 through 2009 we identified 3,717 influenza case-patients. The median age was significantly lower among patients infected with influenza A(H1N1)pdm09 virus than those with seasonal influenza, 17 and 27 years respectively (p<0.001). The vaccine coverage during the influenza season ranged from 3.4% in 2009 to 5.1% in 2006 and was higher in the ≥50 years (range 6.9% in 2008 to 13.2% in 2006) than in the <50 years age group (range 2.2% in 2007 to 3.7% in 2006). The age-adjusted VE estimates for seasonal influenza were 48.6% (4.9%, 73.2%); −14.2% (−9.7%, 34.8%); 12.0% (−70.4%, 55.4%); 67.4% (12.4%, 90.3%) and 29.6% (−21.5%, 60.1%) from 2005 to 2009 respectively. For the A(H1N1)pdm09 season, the efficacy of seasonal vaccine was −6.4% (−93.5%, 43.3%).
Influenza vaccine demonstrated a significant protective effect in two of the five years evaluated. Low vaccine coverage may have reduced power to estimate vaccine effectiveness.
PMCID: PMC3988097  PMID: 24736452
14.  Polymerase activity of hybrid ribonucleoprotein complexes generated from reassortment between 2009 pandemic H1N1 and seasonal H3N2 influenza A viruses 
Virology Journal  2011;8:528.
A novel influenza virus (2009 pdmH1N1) was identified in early 2009 and progressed to a pandemic in mid-2009. This study compared the polymerase activity of recombinant viral ribonucleoprotein (vRNP) complexes derived from 2009 pdmH1N1 and the co-circulating seasonal H3N2, and their possible reassortants.
The 2009 pdmH1N1 vRNP showed a lower level of polymerase activity at 33°C compared to 37°C, a property remenisence of avian viruses. The 2009 pdmH1N1 vRNP was found to be more cold-sensitive than the WSN or H3N2 vRNP. Substituion of 2009 pdmH1N1 vRNP with H3N2-derived-subunits, and vice versa, still retained a substantial level of polymerase activity, which is probably compartable with survival. When the 2009 pdmH1N1 vRNP was substituted with H3N2 PA, a significant increase in activity was observed; whereas when H3N2 vRNP was substituted with 2009 pdmH1N1 PA, a significant decrease in activity occurred. Although, the polymerase basic protein 2 (PB2) of 2009 pdmH1N1 was originated from an avian virus, substitution of this subunit with H3N2 PB2 did not change its polymerase activity in human cells.
In conclusion, our data suggest that hybrid vRNPs resulted from reassortment between 2009 pdmH1N1 and H3N2 viruses could still retain a substantial level of polymerase activity. Substituion of the subunit PA confers the most prominent effect on polymerase activity. Further studies to explore the determinants for polymerase activity of influenza viruses in associate with other factors that limit host specificity are warrant.
PMCID: PMC3267801  PMID: 22152041
Human swine influenza; Pandemic; Seasonal; PB1; PB2; PA; NP; RNP; RNA polymerase; Pathogenesis
15.  Pandemic H1N1 Influenza Infection and Vaccination in Humans Induces Cross-Protective Antibodies that Target the Hemagglutinin Stem 
Most monoclonal antibodies (mAbs) generated from humans infected or vaccinated with the 2009 pandemic H1N1 (pdmH1N1) influenza virus targeted the hemagglutinin (HA) stem. These anti-HA stem mAbs mostly used IGHV1-69 and bound readily to epitopes on the conventional seasonal influenza and pdmH1N1 vaccines. The anti-HA stem mAbs neutralized pdmH1N1, seasonal influenza H1N1 and avian H5N1 influenza viruses by inhibiting HA-mediated fusion of membranes and protected against and treated heterologous lethal infections in mice with H5N1 influenza virus. This demonstrated that therapeutic mAbs could be generated a few months after the new virus emerged. Human immunization with the pdmH1N1 vaccine induced circulating antibodies that when passively transferred, protected mice from lethal, heterologous H5N1 influenza infections. We observed that the dominant heterosubtypic antibody response against the HA stem correlated with the relative absence of memory B cells against the HA head of pdmH1N1, thus enabling the rare heterosubtypic memory B cells induced by seasonal influenza and specific for conserved sites on the HA stem to compete for T-cell help. These results support the notion that broadly protective antibodies against influenza would be induced by successive vaccination with conventional influenza vaccines based on subtypes of HA in viruses not circulating in humans.
PMCID: PMC3347682  PMID: 22586427
pandemic H1N1 influenza; vaccines; cross-protective antibodies; heterosubtypic; plasmablasts; memory B cells; competition for T-cell help; hemagglutinin
16.  Antibody-Dependent Cellular Cytotoxicity Is Associated with Control of Pandemic H1N1 Influenza Virus Infection of Macaques 
Journal of Virology  2013;87(10):5512-5522.
Emerging influenza viruses pose a serious risk to global human health. Recent studies in ferrets, macaques, and humans suggest that seasonal H1N1 (sH1N1) infection provides some cross-protection against 2009 pandemic influenza viruses (H1N1pdm), but the correlates of cross-protection are poorly understood. Here we show that seasonal infection of influenza-naïve Indian rhesus macaques (Macaca mulatta) with A/Kawasaki/173/2001 (sH1N1) virus induces antibodies capable of binding the hemagglutinin (HA) of both the homologous seasonal virus and the antigenically divergent A/California/04/2009 (H1N1pdm) strain in the absence of detectable H1N1pdm-specific neutralizing antibodies. These influenza virus-specific antibodies activated macaque NK cells to express both CD107a and gamma interferon (IFN-γ) in the presence of HA proteins from either sH1N1 or H1N1pdm viruses. Although influenza virus-specific antibody-dependent cellular cytotoxicity (ADCC)-mediated NK cell activation diminished in titer over time following sH1N1 infection, these cells expanded rapidly within 7 days following H1N1pdm exposure. Furthermore, we found that influenza virus-specific ADCC was present in bronchoalveolar lavage fluid and was able to activate lung NK cells. We concluded that infection with a seasonal influenza virus can induce antibodies that mediate ADCC capable of recognizing divergent influenza virus strains. Cross-reactive ADCC may provide a mechanism for reducing the severity of divergent influenza virus infections.
PMCID: PMC3648138  PMID: 23468501
17.  2009 Pandemic Influenza A Virus Subtype H1N1 in Morocco, 2009–2010: Epidemiology, Transmissibility, and Factors Associated With Fatal Cases 
The Journal of Infectious Diseases  2012;206(Suppl 1):S94-S100.
Background. Following the emergence of 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) in the United States and Mexico in April 2009, A(H1N1)pdm09 spread rapidly all over the world. There is a dearth of information about the epidemiology of A(H1N1)pdm09 in Africa, including Morocco. We describe the epidemiologic characteristics of the A(H1N1)pdm09 epidemic in Morocco during 2009–2010, including transmissibility and risk factors associated with fatal disease.
Methods. We implemented influenza surveillance for patients presenting with influenza-like illness (ILI) at 136 private and public clinics for patients with severe acute respiratory illness (SARI) at 16 regional public hospitals from June 2009 through February 2010. Respiratory samples and structured questionnaires were collected from all enrolled patients, and samples were tested by real-time reverse-transcription polymerase chain reaction for influenza viruses. We estimated the risk factors associated with fatal disease as well as the basic reproduction number (R0) and the serial interval of the pandemic virus.
Results. From June 2009 through February 2010, we obtained 3937 specimens, of which 1452 tested positive for influenza virus. Of these, 1398 (96%) were A(H1N1)pdm09. Forty percent of specimens from ILI cases (1056 of 2646) and 27% from SARI cases (342 of 1291) were positive for A(H1N1)pdm09. Sixty-four deaths occurred among laboratory-confirmed A(H1N1)pdm09 SARI cases. Among these cases, those who had hypertension (age-adjusted odd ratio [aOR], 28.2; 95% confidence interval [CI], 2.0–398.7), had neurological disorders (aOR, 7.5; 95% CI, 1.5–36.4), or were obese (aOR, 7.1; 95% CI, 1.6–31.1), as well as women of gestational age who were pregnant (aOR, 2.5; 95% CI, 1.1–5.6), were at increased risk of death. Across the country, elevated numbers of locally acquired infections were detected 4 months after the detection of the first laboratory-confirmed case and coincided with the expected influenza season (October–January) in Morocco. We obtained an R0 estimate of 1.44 (95% CI, 1.32–1.56) and a mean serial interval (±SD) of 2.3 ± 1.4 days (95% CI, 1.6–3.0).
Conclusion. Widespread but delayed community transmission of A(H1N1)pdm09 occurred in Morocco in 2009, and A(H1N1)pdm09 became the dominant influenza virus subtype during the 2009–2010 influenza season. The transmissibility characteristics were similar to those observed in other countries.
PMCID: PMC3572880  PMID: 23169979
18.  Influenza Virus H1N1pdm09 Infections in the Young and Old: Evidence of Greater Antibody Diversity and Affinity for the Hemagglutinin Globular Head Domain (HA1 Domain) in the Elderly than in Young Adults and Children 
Journal of Virology  2012;86(10):5515-5522.
The H1N1 2009 influenza virus (H1N1pdm09) pandemic had several unexpected features, including low morbidity and mortality in older populations. We performed in-depth evaluation of antibody responses generated following H1N1pdm09 infection of naïve ferrets and of 130 humans ranging from the very young (0 to 9 years old) to the very old (70 to 89 years old). In addition to hemagglutination inhibition (HI) titers, we used H1N1pdm09 whole-genome-fragment phage display libraries (GFPDL) to evaluate the antibody repertoires against internal genes, hemagglutinin (HA), and neuraminidase (NA) and also measured antibody affinity for antigenic domains within HA. GFPDL analyses of H1N1pdm09-infected ferrets demonstrated gradual development of antibody repertoires with a focus on M1 and HA1 by day 21 postinfection. In humans, H1N1pdm09 infection in the elderly (>70 years old) induced antibodies with broader epitope recognition in both the internal genes and the HA1 receptor binding domain (RBD) than for the younger age groups (0 to 69 years). Importantly, post-H1N1 infection serum antibodies from the elderly demonstrated substantially higher avidity for recombinant HA1 (rHA1) (but not HA2) than those from younger subjects (50% versus <22% 7 M urea resistance, respectively) and lower antibody dissociation rates using surface plasmon resonance. This is the first study in humans that provides evidence for a qualitatively superior antibody response in the elderly following H1N1pdm09 infection, indicative of recall of long-term memory B cells or long-lived plasma cells. These findings may help explain the age-related morbidity and mortality pattern observed during the H1N1pdm09 pandemic.
PMCID: PMC3347288  PMID: 22379097
19.  H1N1pdm Influenza Infection in Hospitalized Cancer Patients: Clinical Evolution and Viral Analysis 
PLoS ONE  2010;5(11):e14158.
The novel influenza A pandemic virus (H1N1pdm) caused considerable morbidity and mortality worldwide in 2009. The aim of the present study was to evaluate the clinical course, duration of viral shedding, H1N1pdm evolution and emergence of antiviral resistance in hospitalized cancer patients with severe H1N1pdm infections during the winter of 2009 in Brazil.
We performed a prospective single-center cohort study in a cancer center in Rio de Janeiro, Brazil. Hospitalized patients with cancer and a confirmed diagnosis of influenza A H1N1pdm were evaluated. The main outcome measures in this study were in-hospital mortality, duration of viral shedding, viral persistence and both functional and molecular analyses of H1N1pdm susceptibility to oseltamivir.
A total of 44 hospitalized patients with suspected influenza-like illness were screened. A total of 24 had diagnosed H1N1pdm infections. The overall hospital mortality in our cohort was 21%. Thirteen (54%) patients required intensive care. The median age of the studied cohort was 14.5 years (3–69 years). Eighteen (75%) patients had received chemotherapy in the previous month, and 14 were neutropenic at the onset of influenza. A total of 10 patients were evaluated for their duration of viral shedding, and 5 (50%) displayed prolonged viral shedding (median 23, range = 11–63 days); however, this was not associated with the emergence of a resistant H1N1pdm virus. Viral evolution was observed in sequentially collected samples.
Prolonged influenza A H1N1pdm shedding was observed in cancer patients. However, oseltamivir resistance was not detected. Taken together, our data suggest that severely ill cancer patients may constitute a pandemic virus reservoir with major implications for viral propagation.
PMCID: PMC2994772  PMID: 21152402
20.  Phenotypic Differences in Virulence and Immune Response in Closely Related Clinical Isolates of Influenza A 2009 H1N1 Pandemic Viruses in Mice 
PLoS ONE  2013;8(2):e56602.
To capture the possible genotypic and phenotypic differences of the 2009 influenza A virus H1N1 pandemic (H1N1pdm) strains circulating in adult hospitalized patients, we isolated and sequenced nine H1N1pdm viruses from patients hospitalized during 2009–2010 with severe influenza pneumonia in Kentucky. Each viral isolate was characterized in mice along with two additional H1N1 pandemic strains and one seasonal strain to assess replication and virulence. All isolates showed similar levels of replication in nasal turbinates and lung, but varied in their ability to cause morbidity. Further differences were identified in cytokine and chemokine responses. IL-6 and KC were expressed early in mice infected with strains associated with higher virulence. Strains that showed lower pathogenicity in mice had greater IFNγ, MIG, and IL-10 responses. A principal component analysis (PCA) of the cytokine and chemokine profiles revealed 4 immune response phenotypes that correlated with the severity of disease. A/KY/180/10, which showed the greatest virulence with a rapid onset of disease progression, was compared in additional studies with A/KY/136/09, which showed low virulence in mice. Analyses comparing a low (KY/136) versus a high (KY/180) virulent isolate showed a significant difference in the kinetics of infection within the lower respiratory tract and immune responses. Notably by 4 DPI, virus titers within the lung, bronchoalveolar lavage fluid (BALf), and cells within the BAL (BALc) revealed that the KY/136 replicated in BALc, while KY/180 replication persisted in lungs and BALc. In summary, our studies suggest four phenotypic groups based on immune responses that result in different virulence outcomes in H1N1pdm isolates with a high degree of genetic similarity. In vitro studies with two of these isolates suggested that the more virulent isolate, KY/180, replicates productively in macrophages and this may be a key determinant in tipping the response toward a more severe disease progression.
PMCID: PMC3575477  PMID: 23441208
21.  Interleukin-6 Is a Potential Biomarker for Severe Pandemic H1N1 Influenza A Infection 
PLoS ONE  2012;7(6):e38214.
Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data.
PMCID: PMC3367995  PMID: 22679491
22.  Genetic and Phylogenetic Analyses of Influenza A H1N1pdm Virus in Buenos Aires, Argentina ▿ †  
Journal of Virology  2010;85(2):1058-1066.
An influenza pandemic caused by swine-origin influenza virus A/H1N1 (H1N1pdm) spread worldwide in 2009, with 12,080 confirmed cases and 626 deaths occurring in Argentina. A total of 330 H1N1pdm viruses were detected from May to August 2009, and phylogenetic and genetic analyses of 21 complete genome sequences from both mild and fatal cases were achieved with reference to concatenated whole genomes. In addition, the analysis of another 16 hemagglutinin (HA), neuraminidase (NA), and matrix (M) gene sequences of Argentinean isolates was performed. The microevolution timeline was assessed and resistance monitoring of an NA fragment from 228 samples throughout the 2009 pandemic peak was performed by sequencing and pyrosequencing. We also assessed the viral growth kinetics for samples with replacements at the genomic level or special clinical features. In this study, we found by Bayesian inference that the Argentinean complete genome sequences clustered with globally distributed clade 7 sequences. The HA sequences were related to samples from the northern hemisphere autumn-winter from September to December 2009. The NA of Argentinean sequences belonged to the New York group. The N-4 fragment as well as the hierarchical clustering of samples showed that a consensus sequence prevailed in time but also that different variants, including five H275Y oseltamivir-resistant strains, arose from May to August 2009. Fatal and oseltamivir-resistant isolates had impaired growth and a small plaque phenotype compared to oseltamivir-sensitive and consensus strains. Although these strains might not be fit enough to spread in the entire population, molecular surveillance proved to be essential to monitor resistance and viral dynamics in our country.
PMCID: PMC3020011  PMID: 21047959
23.  Estimating age-specific influenza-related hospitalization rates during the pandemic (H1N1) 2009 in Davidson Co, TN 
In April 2009, a pandemic caused by a novel influenza strain, the A(H1N1)pdm09 virus, started. Few age-specific estimates of hospitalizations associated with the first year of circulation of the pandemic virus are available.
To estimate age-specific hospitalization rates associated with laboratory-confirmed A(H1N1)pdm09 virus in Davidson County, TN, from May 2009 to March 2010.
Two separate strategies were applied: capture–recapture and surveillance-sampling methods. For the capture–recapture estimates, we linked data collected via two independent prospective population-based surveillance systems: The Influenza Vaccine Effectiveness Network (Flu-VE) tested consenting county patients hospitalized with respiratory symptoms at selected hospitals using real-time reverse transcriptase polymerase chain reaction (rRT-PCR); the Emerging Infections Program identified county patients with positive influenza tests in all area hospitals. For the surveillance-sampling estimates, we applied the agespecific proportions of influenza-positive patients (from Flu-VE) to the number of acute respiratory illness hospitalizations obtained from the Tennessee Hospital Discharge Data system.
With capture–recapture, we estimated 0.89 (95% CI, 0.72–1.49), 0.62 (0.42–1.11), 1.78 (0.99–3.63), and 0.76 (0.50–1.76) hospitalizations per 1000 residents aged <5, 5–17, 18–49, and ≥50 years, respectively. Surveillance-sampling estimated rates were 0.78 (0.46–1.22), 0.32 (0.14–0.69), 0.99 (0.64–1.52), and 1.43 (0.80–2.48) hospitalizations per 1000 residents aged <5, 5–17, 18–49, and ≥50 years, respectively. In all age-groups combined, we estimated approximately 1 influenza-related hospitalization per 1000 residents.
Two independent methods provided consistent results on the burden of pandemic virus in Davidson County and suggested that the overall incidence of A(H1N1)pdm09-associated hospitalization was 1 per 1000 county residents.
PMCID: PMC3773818  PMID: 22360812
A(H1N1)pdm09 virus; capture–recapture; hospitalization rates; surveillance-sampling
24.  Transmission Characteristics of the 2009 H1N1 Influenza Pandemic: Comparison of 8 Southern Hemisphere Countries 
PLoS Pathogens  2011;7(9):e1002225.
While in Northern hemisphere countries, the pandemic H1N1 virus (H1N1pdm) was introduced outside of the typical influenza season, Southern hemisphere countries experienced a single wave of transmission during their 2009 winter season. This provides a unique opportunity to compare the spread of a single virus in different countries and study the factors influencing its transmission. Here, we estimate and compare transmission characteristics of H1N1pdm for eight Southern hemisphere countries/states: Argentina, Australia, Bolivia, Brazil, Chile, New Zealand, South Africa and Victoria (Australia). Weekly incidence of cases and age-distribution of cumulative cases were extracted from public reports of countries' surveillance systems. Estimates of the reproduction numbers, R0, empirically derived from the country-epidemics' early exponential phase, were positively associated with the proportion of children in the populations (p = 0.004). To explore the role of demography in explaining differences in transmission intensity, we then fitted a dynamic age-structured model of influenza transmission to available incidence data for each country independently, and for all the countries simultaneously. Posterior median estimates of R0 ranged 1.2–1.8 for the country-specific fits, and 1.29–1.47 for the global fits. Corresponding estimates for overall attack-rate were in the range 20–50%. All model fits indicated a significant decrease in susceptibility to infection with age. These results confirm the transmissibility of the 2009 H1N1 pandemic virus was relatively low compared with past pandemics. The pattern of age-dependent susceptibility found confirms that older populations had substantial – though partial - pre-existing immunity, presumably due to exposure to heterologous influenza strains. Our analysis indicates that between-country-differences in transmission were at least partly due to differences in population demography.
Author Summary
Although relatively mild, the 2009 H1N1 pandemic reminded us once again of the on-going threat posed by novel respiratory viruses and the need for understanding better how such pathogens emerge and spread. From April to September 2009, countries in temperate regions of the Southern hemisphere experienced large epidemics of H1N1pdm during their winter season, with the new virus quickly becoming the predominant circulating influenza strain. We use mathematical modelling to analyse H1N1pdm epidemiological data from 8 southern hemisphere countries. We aim at understanding better the factors which may have influenced virus transmission in these countries. We find that transmissibility of the virus was relatively low compared with previous influenza pandemics, largely because of strong pre-existing age-dependent susceptibility to the virus (older people being less susceptible to infection, perhaps due to pre-existing immunity). We suggest that population demography had a strong impact on the virus spread and that higher transmission rates occurred in countries having a younger population. Our results highlight the requirement to use age-structured models for the analysis of influenza epidemics and support the need for country-specific analyses to inform the design of control policies for pandemic mitigation.
PMCID: PMC3164643  PMID: 21909272
25.  H1N1pdm in the Americas 
Epidemics  2010;2(3):132-138.
In late April 2009 the emergence of 2009 pandemic influenza A (H1N1pdm) virus was detected in humans. From its detection through July 18th, 2009, confirmed cases of H1N1pdm in the Americas were periodically reported to the Pan-American Health Organization (PAHO) by member states. Because the Americas span much of the world’s latitudes, this data provides an excellent opportunity to examine variation in H1N1pdm transmission by season.
Using reports from PAHO member states from April 26th, 2009 through July 18th, 2009, we characterize the early spread of the H1N1 pandemic in the Americas. For a geographically representative sample of member states we estimate the reproductive number (R) of H1N1pdm over the reporting period. The association between these estimates and latitude, temperature, humidity and population age structure was estimated.
Estimates of the peak reproductive number of H1N1pdm ranged from 1.3 (for Panama, Colombia) to 2.1 (for Chile). We found that reproductive number estimates were most associated with latitude in both univariate and multivariate analyses. To the extent that latitude is a proxy for seasonal changes in climate and behavior, this association suggests a strong seasonal component to H1N1pdm transmission. However, the reasons for this seasonality remain unclear.
PMCID: PMC2937282  PMID: 20847900
pandemic H1N1; influenza; seasonality; reproductive number

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