Effect of priming with H1N1 influenza viruses of variable antigenic distances on challenge with 2009 pandemic H1N1 virus

Compared to seasonal influenza viruses, the 2009 pandemic H1N1 (pH1N1) virus caused greater morbidity and mortality in children and young adults. People over 60 years of age showed a higher prevalence of cross-reactive pH1N1 antibodies, suggesting that they were previously exposed to an influenza virus or vaccine that was antigenically related to the pH1N1 virus. To define the basis for this cross-reactivity, ferrets were infected with H1N1 viruses of variable antigenic distance that circulated during different decades from the 1930s (Alaska/35), 1940s (Fort Monmouth/47), 1950s (Fort Warren/50), and 1990s (New Caledonia/99) and challenged with 2009 pH1N1 virus 6 weeks later. Ferrets primed with the homologous CA/09 or New Jersey/76 (NJ/76) virus served as a positive control, while the negative control was an influenza B virus that should not cross-protect against influenza A virus infection. Significant protection against challenge virus replication in the respiratory tract was observed in ferrets primed with AK/35, FM/47, and NJ/76; FW/50-primed ferrets showed reduced protection, and NC/99-primed ferrets were not protected. The hemagglutinins (HAs) of AK/35, FM/47, and FW/50 differ in the presence of glycosylation sites. We found that the loss of protective efficacy observed with FW/50 was associated with the presence of a specific glycosylation site. Our results suggest that changes in the HA occurred between 1947 and 1950, such that prior infection could no longer protect against 2009 pH1N1 infection. This provides a mechanistic understanding of the nature of serological cross-protection observed in people over 60 years of age during the 2009 H1N1 pandemic.     

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.     

Comparative analyses of pandemic H1N1 and seasonal H1N1, H3N2, and influenza B infections depict distinct clinical pictures in ferrets

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.     

Replication and pathogenesis of the pandemic (H1N1) 2009 influenza virus in mammalian models

This study aimed to characterize the replication and pathogenic properties of a Korean pandemic (H1N1) 2009 influenza virus isolate in ferrets and mice. Ferrets infected with A/Korea/01/2009 (H1N1) virus showed mild clinical signs. The virus replicated well in lungs and slightly in brains with no replication in any other organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected in the bronchiolar epithelial cells, in peribronchial glands with severe peribronchitis and in cells present in the alveoli. A/Korea/01/2009 (H1N1) virus-infected mice showed weight loss and pathological lung lesions including perivascular cuffing, interstitial pneumonia and alveolitis. The virus replicated highly in the lungs and slightly in the nasal tissues. Viral antigens were detected in bronchiolar epithelial cells, pneumocytes and interstitial macrophages. However, seasonal H1N1 influenza virus did not replicate in the lungs of ferrets, and viral antigens were not detected. Thus, this Korean pandemic (H1N1) 2009 isolate infected the lungs of ferrets and mice successfully and caused more pathological lesions than did the seasonal influenza virus.     

Pathogenesis and transmission of triple-reassortant swine H1N1 influenza viruses isolated before the 2009 H1N1 pandemic

The 2009 H1N1 pandemic influenza virus represents the greatest incidence of human infection with an influenza virus of swine origin to date. Moreover, triple-reassortant swine (TRS) H1N1 viruses, which share similar host and lineage origins with 2009 H1N1 viruses, have been responsible for sporadic human cases since 2005. Similar to 2009 H1N1 viruses, TRS viruses are capable of causing severe disease in previously healthy individuals and frequently manifest with gastrointestinal symptoms; however, their ability to cause severe disease has not been extensively studied. Here, we evaluated the pathogenicity and transmissibility of two TRS viruses associated with disease in humans in the ferret model. TRS and 2009 H1N1 viruses exhibited comparable viral titers and histopathologies following virus infection and were similarly unable to transmit efficiently via respiratory droplets in the ferret model. Utilizing TRS and 2009 H1N1 viruses, we conducted extensive hematologic and blood serum analyses on infected ferrets to identify lymphohematopoietic parameters associated with mild to severe influenza virus infection. Following H1N1 or H5N1 influenza virus infection, ferrets were found to recapitulate several laboratory abnormalities previously documented with human disease, furthering the utility of the ferret model for the assessment of influenza virus pathogenicity.     

Eurasian-origin gene segments contribute to the transmissibility, aerosol release, and morphology of the 2009 pandemic H1N1 influenza virus

The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus.     

Impact of Prior Seasonal H3N2 Influenza Vaccination or Infection on Protection and Transmission of Emerging Variants of Influenza A(H3N2)v Virus in Ferrets

Influenza H3N2 A viruses continue to circulate in swine and occasionally infect humans, resulting in outbreaks of variant influenza H3N2 [A(H3N2)v] virus. It has been previously demonstrated in ferrets that A(H3N2)v viruses transmit as efficiently as seasonal influenza viruses, raising concern over the pandemic potential of these viruses. However, A(H3N2)v viruses have not acquired the ability to transmit efficiently among humans, which may be due in part to existing cross-reactive immunity to A(H3N2)v viruses. Although current seasonal H3N2 and A(H3N2)v viruses are antigenically distinct from one another, historical H3N2 viruses have some antigenic similarity to A(H3N2)v viruses and previous exposure to these viruses may provide a measure of immune protection sufficient to dampen A(H3N2)v virus transmission. Here, we evaluated whether prior seasonal H3N2 influenza virus vaccination or infection affects virus replication and transmission of A(H3N2)v virus in the ferret animal model. We found that the seasonal trivalent inactivated influenza virus vaccine (TIV) or a monovalent vaccine prepared from an antigenically related 1992 seasonal influenza H3N2 (A/Beijing/32/1992) virus failed to substantially reduce A(H3N2)v (A/Indiana/08/2011) virus shedding and subsequent transmission to naive hosts. Conversely, ferrets primed by seasonal H3N2 virus infection displayed reduced A(H3N2)v virus shedding following challenge, which blunted transmission to naive ferrets. A higher level of specific IgG and IgA antibody titers detected among infected versus vaccinated ferrets was associated with the degree of protection offered by seasonal H3N2 virus infection. The data demonstrate in ferrets that the efficiency of A(H3N2)v transmission is disrupted by preexisting immunity induced by seasonal H3N2 virus infection.     

Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.     

Influenza A: From highly pathogenic H5N1 to pandemic 2009 H1N1. Epidemiology and clinical features

The last decade has seen the emergence of two new influenza A subtypes and they have become a cause of concern for the global community. These are the highly pathogenic H5N1 influenza A virus (H5N1) and the Pandemic 2009 influenza H1N1 virus. Since 2003 the H5N1 virus has caused widespread disease and death in poultry, mainly in south East Asia and Africa. In humans the number of cases infected with this virus is few but the mortality has been about 60%. Most patients have presented with severe pneumonia and acute respiratory distress syndrome. The second influenza virus, the pandemic H1N1 2009, emerged in Mexico in March this year. This virus acquired the ability for sustained human to human spread and within a few months spread throughout the world and infected over 4 lakh individuals. The symptoms of infection with this virus are similar to seasonal influenza but it currently affecting younger individuals more often. Fortunately the mortality has been low. Both these new influenza viruses are currently circulating and have different clinical and epidemiological characteristics.     

Matrix-M Adjuvated Seasonal Virosomal Influenza Vaccine Induces Partial Protection in Mice and Ferrets against Avian H5 and H7 Challenge

There is a constant threat of zoonotic influenza viruses causing a pandemic outbreak in humans. It is virtually impossible to predict which virus strain will cause the next pandemic and it takes a considerable amount of time before a safe and effective vaccine will be available once a pandemic occurs. In addition, development of pandemic vaccines is hampered by the generally poor immunogenicity of avian influenza viruses in humans. An effective pre-pandemic vaccine is therefore required as a first line of defense. Broadening of the protective efficacy of current seasonal vaccines by adding an adjuvant may be a way to provide such first line of defense. Here we evaluate whether a seasonal trivalent virosomal vaccine (TVV) adjuvated with the saponin-based adjuvant Matrix-M (MM) can confer protection against avian influenza H5 and H7 virus strains in mice and ferrets. We demonstrate that mice were protected from death against challenges with H5N1 and H7N7, but that the protection was not complete as evidenced by severe clinical signs. In ferrets, protection against H7N9 was not observed. In contrast, reduced upper and lower respiratory tract viral loads and reduced lung pathology, was achieved in H5N1 challenged ferrets. Together these results suggest that, at least to some extent, Matrix-M adjuvated seasonal virosomal influenza vaccine can serve as an interim measure to decrease morbidity and mortality associated with a pandemic outbreak.     

Systemic dissemination of H5N1 influenza A viruses in ferrets and hamsters after direct intragastric inoculation

Although oral exposure to H5N1 highly pathogenic avian influenza viruses is a risk factor for infection in humans, it is unclear how oral exposure to these virus results in lethal respiratory infections. To address this issue, we inoculated ferrets and hamsters with two highly pathogenic H5N1 strains. These viruses, inoculated directly into the stomach, were isolated from the large intestine and the mesenteric lymph nodes within 1 day of inoculation and subsequently spread to multiple tissues, including lung, liver, and brain. Histopathologic analysis of ferrets infected with virus via direct intragastric inoculation revealed lymph folliculitis in the digestive tract and mesenteric lymph nodes and focal interstitial pneumonia. Comparable results were obtained with the hamster model. We conclude that, in mammals, ingested H5N1 influenza viruses can disseminate to nondigestive organs, possibly through the lymphatic system of the gastrointestinal tract.     

In Vivo Selection of H1N2 Influenza Virus Reassortants in the Ferret Model

Although the ferret model has been extensively used to study pathogenesis and transmission of influenza viruses, little has been done to determine whether ferrets are a good surrogate animal model to study influenza virus reassortment. It has been previously shown that the pandemic 2009 H1N1 (H1N1pdm) virus was able to transmit efficiently in ferrets. In coinfection studies with either seasonal H1N1 or H3N2 strains (H1N1s or H3N2s, respectively), the H1N1pdm virus was able to outcompete these strains and become the dominant transmissible virus. However, lack of reassortment could have been the result of differences in the cell or tissue tropism of these viruses in the ferret. To address this issue, we performed coinfection studies with recombinant influenza viruses carrying the surface genes of a seasonal H3N2 strain in the background of an H1N1pdm strain and vice versa. After serial passages in ferrets, a dominant H1N2 virus population was obtained with a constellation of gene segments, most of which, except for the neuraminidase (NA) and PB1 segments, were from the H1N1pdm strain. Our studies suggest that ferrets recapitulate influenza virus reassortment events. The H1N2 virus generated through this process resembles similar viruses that are emerging in nature, particularly in pigs.     

Characterization of the 2009 Pandemic A/Beijing/501/2009 H1N1 Influenza Strain in Human Airway Epithelial Cells and Ferrets

Background

A novel 2009 swine-origin influenza A H1N1 virus (S-OIV H1N1) has been transmitted among humans worldwide. However, the pathogenesis of this virus in human airway epithelial cells and mammals is not well understood.

Methodology/Principal Finding

In this study, we showed that a 2009 A (H1N1) influenza virus strain, A/Beijing/501/2009, isolated from a human patient, caused typical influenza-like symptoms including weight loss, fluctuations in body temperature, and pulmonary pathological changes in ferrets. We demonstrated that the human lung adenocarcinoma epithelial cell line A549 was susceptible to infection and that the infected cells underwent apoptosis at 24 h post-infection. In contrast to the seasonal H1N1 influenza virus, the 2009 A (H1N1) influenza virus strain A/Beijing/501/2009 induced more cell death involving caspase-3-dependent apoptosis in A549 cells. Additionally, ferrets infected with the A/Beijing/501/2009 H1N1 virus strain exhibited increased body temperature, greater weight loss, and higher viral titers in the lungs. Therefore, the A/Beijing/501/2009 H1N1 isolate successfully infected the lungs of ferrets and caused more pathological lesions than the seasonal influenza virus. Our findings demonstrate that the difference in virulence of the 2009 pandemic H1N1 influenza virus and the seasonal H1N1 influenza virus in vitro and in vivo may have been mediated by different mechanisms.

Conclusion/Significance

Our understanding of the pathogenesis of the 2009 A (H1N1) influenza virus infection in both humans and animals is broadened by our findings that apoptotic cell death is involved in the cytopathic effect observed in vitro and that the pathological alterations in the lungs of S-OIV H1N1-infected ferrets are much more severe.     

The Potential of Avian H1N1 Influenza A Viruses to Replicate and Cause Disease in Mammalian Models

H1N1 viruses in which all gene segments are of avian origin are the most frequent cause of influenza pandemics in humans; therefore, we examined the disease-causing potential of 31 avian H1N1 isolates of American lineage in DBA/2J mice. Thirty of 31 isolates were very virulent, causing respiratory tract infection; 22 of 31 resulted in fecal shedding; and 10 of 31 were as pathogenic as the pandemic 2009 H1N1 viruses. Preliminary studies in BALB/cJ mice and ferrets showed that 1 of 4 isolates tested was more pathogenic than the pandemic 2009 H1N1 viruses in BALB/cJ mice, and 1 of 2 strains transmitted both by direct and respiratory-droplet contact in ferrets. Preliminary studies of other avian subtypes (H2, H3, H4, H6, H10, H12) in DBA/2J mice showed lower pathogenicity than the avian H1N1 viruses. These findings suggest that avian H1N1 influenza viruses are unique among influenza A viruses in their potential to infect mammals.     

Pandemic H1N1 2009 Influenza A Virus Induces Weak Cytokine Responses in Human Macrophages and Dendritic Cells and Is Highly Sensitive to the Antiviral Actions of Interferons

In less than 3 months after the first cases of swine origin 2009 influenza A (H1N1) virus infections were reported from Mexico, WHO declared a pandemic. The pandemic virus is antigenically distinct from seasonal influenza viruses, and the majority of human population lacks immunity against this virus. We have studied the activation of innate immune responses in pandemic virus-infected human monocyte-derived dendritic cells (DC) and macrophages. Pandemic A/Finland/553/2009 virus, representing a typical North American/European lineage virus, replicated very well in these cells. The pandemic virus, as well as the seasonal A/Brisbane/59/07 (H1N1) and A/New Caledonia/20/99 (H1N1) viruses, induced type I (alpha/beta interferon [IFN-α/β]) and type III (IFN-λ1 to -λ3) IFN, CXCL10, and tumor necrosis factor alpha (TNF-α) gene expression weakly in DCs. Mouse-adapted A/WSN/33 (H1N1) and human A/Udorn/72 (H3N2) viruses, instead, induced efficiently the expression of antiviral and proinflammatory genes. Both IFN-α and IFN-β inhibited the replication of the pandemic (H1N1) virus. The potential of IFN-λ3 to inhibit viral replication was lower than that of type I IFNs. However, the pandemic virus was more sensitive to the antiviral IFN-λ3 than the seasonal A/Brisbane/59/07 (H1N1) virus. The present study demonstrates that the novel pandemic (H1N1) influenza A virus can readily replicate in human primary DCs and macrophages and efficiently avoid the activation of innate antiviral responses. It is, however, highly sensitive to the antiviral actions of IFNs, which may provide us an additional means to treat severe cases of infection especially if significant drug resistance emerges.The novel swine origin 2009 influenza A (H1N1) virus was identified in April 2009, and it is currently causing the first influenza pandemic of the 21st century. The virus is a completely new reassortant virus (8, 38), and the majority of the human population does not have preexisting immunity against it. The case fatality rate of the current pandemic virus infection is still unclear, but it is estimated to be somewhat higher than that of seasonal influenza virus infections (8). In most cases, the pandemic 2009 A (H1N1) virus causes an uncomplicated respiratory tract illness with symptoms similar to those caused by seasonal influenza viruses. However, gastrointestinal symptoms atypical to seasonal influenza have been detected in a significant proportion of cases (4, 7, 35).The pandemic 2009 (H1N1) influenza A virus originates from a swine influenza A virus strain. It underwent multiple reassortment events in pigs and then transferred into the human population (8, 38). The new virus has gene segments from the North American triple-reassortant and Eurasian swine H1N1 viruses (8, 38). Sequence analysis of this new pandemic virus revealed that hemagglutinin (HA), NP, and NS gene segments are derived from the classical swine viruses, PB1 from human H3N2, and PB2 and PA from avian viruses within the triple-reassortant virus (8). In addition, the NA and M segments originate from the Eurasian swine virus lineage. The pandemic 2009 (H1N1) virus is genetically and antigenically distinct from previous seasonal human influenza A (H1N1) viruses. Thus, the current seasonal influenza vaccines are likely to give little, if any, protection against pandemic 2009 A (H1N1) virus infection (12, 14). However, some evidence indicates that people born early in the 20th century have cross-neutralizing antibodies against the pandemic 2009 A (H1N1) viruses (12, 14).At present, relatively little is known about the pathogenesis and transmission of the pandemic 2009 A (H1N1) virus in humans. Studies with ferrets revealed that the pandemic virus replicated better than seasonal H1N1 viruses in the respiratory tracts of the animals. This suggests that the virus is more pathogenic in ferrets than seasonal influenza viruses (19, 24). The respiratory tract is the primary infection site of all mammalian influenza viruses, and, indeed, the specific glycan receptors on the apical surface of the upper respiratory tract have been reported to bind HA of the 2009 A (H1N1) virus (19). In human lung tissue binding assays, 2009 A (H1N1) HA showed a glycan binding pattern similar to that of the HA from the pandemic 1918 A (H1N1) virus though its affinity to α2,6 glycans was much lower than that of the 1918 virus HA. The lower glycan binding properties of the pandemic 2009 A (H1N1) virus seemed to correlate with less-efficient transmission in ferrets compared to seasonal H1N1 viruses (19). According to another study with ferrets, the transmission of the pandemic 2009 A (H1N1) virus via respiratory droplets was as efficient as that of a seasonal A (H1N1) virus (24). It is clear that, besides experimental infections in animal models, analyses of the characters and pathogenesis of the pandemic 2009 A (H1N1) virus infection in humans are urgently needed.In the present study, we have focused on analyzing innate immune responses in primary human dendritic cells (DCs) and macrophages in response to an infection with one of the Finnish isolates of the pandemic 2009 A (H1N1) virus. DCs and macrophages reside beneath the epithelium of the respiratory organs, and these cells are thus potential targets for influenza viruses. From the epithelial cells influenza viruses spread in DCs and macrophages, which coordinate the development of an effective innate immune response against the virus (22, 34, 41). During influenza virus infection, DCs and macrophages secrete antiviral cytokines such as interferons (IFNs) and tumor necrosis factor alpha (TNF-α) (3, 13, 26). Moreover, DCs and macrophages activate virus-destroying NK cells and T cells with the cytokines they secrete and via direct cell-to-cell contacts (9, 29, 33, 37). Here we show that the pandemic (H1N1) virus infects and replicates very well in human monocyte-derived DCs and macrophages. The pandemic virus as well as two recent seasonal H1N1 viruses induced a relatively weak innate immune response in these cells, as evidenced by a poor expression of antiviral and proinflammatory cytokine genes. However, like seasonal influenza A viruses, the pandemic 2009 (H1N1) virus was extremely sensitive to the antiviral actions of type I IFNs (IFN-α/β). Interestingly, the pandemic 2009 (H1N1) virus was even more sensitive to antiviral IFN-λ3 than a seasonal A (H1N1) virus. Thus, IFNs may provide us with an additional means to combat severe pandemic influenza virus infections, especially if viral resistance against neuraminidase (NA) inhibitors begins to emerge.     

Age-matched comparison of children hospitalized for 2009 pandemic H1N1 influenza with those hospitalized for seasonal H1N1 and H3N2

BACKGROUND: A wide spectrum of clinical manifestation ranging from deaths to a mild course of disease has been reported in children infected with the 2009 pandemic H1N1 (pH1N1) influenza. METHODOLOGY/MAJOR FINDINGS: We conducted an age-matched control study comparing children hospitalized for pH1N1 with historic controls infected with seasonal H1N1 and H3N2 influenza to correct for the effect of age on disease susceptibility and clinical manifestations. We also compared children with pH1N1 to children concurrently admitted for seasonal influenza during the pandemic period to adjust for differences in health-seeking behavior during the pandemic or other potential bias associated with historic controls. There was no death or intensive care admission. Children with pH1N1 were more likely to have at least one risk condition for influenza, an underlying chronic pulmonary condition, more likely to have asthma exacerbation and to be treated with oseltamivir. There was no difference in other aspects of the clinical course or outcome. CONCLUSION: Disease manifestation of children hospitalized for pH1N1 infection was mild in our patient population.     

Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.     

Replication and Immunogenicity of Swine,Equine, and Avian H3 Subtype Influenza Viruses in Mice and Ferrets

Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines.     

Recently emerged swine influenza A virus (H2N3) causes severe pneumonia in Cynomolgus macaques

The triple reassortant H2N3 virus isolated from diseased pigs in the United States in 2006 is pathogenic for certain mammals without prior adaptation and transmits among swine and ferrets. Adaptation, in the H2 hemagglutinin derived from an avian virus, includes the ability to bind to the mammalian receptor, a significant prerequisite for infection of mammals, in particular humans, which poses a big concern for public health. Here we investigated the pathogenic potential of swine H2N3 in Cynomolgus macaques, a surrogate model for human influenza infection. In contrast to human H2N2 virus, which served as a control and largely caused mild pneumonia similar to seasonal influenza A viruses, the swine H2N3 virus was more pathogenic causing severe pneumonia in nonhuman primates. Both viruses replicated in the entire respiratory tract, but only swine H2N3 could be isolated from lung tissue on day 6 post infection. All animals cleared the infection whereas swine H2N3 infected macaques still presented with pathologic changes indicative of chronic pneumonia at day 14 post infection. Swine H2N3 virus was also detected to significantly higher titers in nasal and oral swabs indicating the potential for animal-to-animal transmission. Plasma levels of IL-6, IL-8, MCP-1 and IFNγ were significantly increased in swine H2N3 compared to human H2N2 infected animals supporting the previously published notion of increased IL-6 levels being a potential marker for severe influenza infections. In conclusion, the swine H2N3 virus represents a threat to humans with the potential for causing a larger outbreak in a non-immune or partially immune population. Furthermore, surveillance efforts in farmed pig populations need to become an integral part of any epidemic and pandemic influenza preparedness.