Swine H1N1 Viruses Research Articles

Characterization of an Artificial Swine-Origin Influenza Virus with the Same Gene Combination as H1N1/2009 Virus: A Genesis Clue of Pandemic Strain

Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.

Serological and Virological Surveillance of Swine H1N1 and H3N2 Influenza Virus Infection in Two Farms Located in Hubei Province, Central China

Swine influenza viruses H1N1 and H3N2 have been reported in the swine population worldwide. From June 2008 to June 2009, we carried out serological and virological surveillance of swine influenza in the Hubei province in central China. The serological results indicated that antibodies to H1N1 swine influenza virus in the swine population were high with a 42.5% (204/480) positive rate, whereas antibodies to H3N2 swine influenza virus were low with a 7.9% (38/480) positive rate. Virological surveillance showed that only one sample from weanling pigs was positive by RT-PCR. Phylogenetic analysis of the hemagglutinin and neuraminidase genes revealed that the A/Sw/HB/S1/2009 isolate was closely related to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses. In conclusion, H1N1 influenza viruses were more dominant in the pig population than H3N2 influenza viruses in central China, and infection with avian-like H1N1 viruses persistently emerged in the swine population in the area.

Emergence of novel reassortant H3N2 influenza viruses among ducks in China

During 2006-2009 influenza virus surveillance, three H3N2 viruses were isolated from ducks in Central China. Sequence and phylogenetic analyses revealed that most segments of these three isolates had high identity with H3N2 swine isolates in South China. However, for M, the three viruses, along with H1N1 swine isolates of North America, formed a cluster; for PB2, two of these isolates fell into the cluster of the H5N1 duck isolates, indicating a reassortment among H3N2, H1N1 swine viruses and H5N1 avian virus. The emergence of H3N2 virus with incorporation of an H5N1 virus gene raises new concerns about the generation of novel viruses that could affect humans.

Perspective: Swine-Origin Influenza: 1976 and 2009

I am in a unique situation, having been involved in 2 major US public health events resulting from novel swine-origin influenza viruses. In 1976, I was Director of the Center for Disease Control (CDC, the name of the agency at the time) when a new influenza virus, characterized as an influenza A(H1N1) swine virus, was isolated from military recruits at Fort Dix, New Jersey. Subsequently, I led the CDC through the US response to this outbreak, which culminated in the decision to implement the swine flu vaccination program during which 45 million people were vaccinated over 10 weeks. The program was stopped after cases of Guillain-Barré Syndrome were identified following vaccination and when no spread of the virus occurred beyond Fort Dix. In 2009, as another new swine H1N1 virus was first identified and emergency response began, I was asked to be an advisor to the CDC Director in order that I might provide historical context to the novel H1N1 swine-origin outbreak and response. In this latter capacity, I have been able to observe and participate in discussions resulting in decision-making for the CDC's national response to this public health emergency as an unpaid consultant. This paper is a personal commentary on the similarities and dissimilarities of the 2 episodes.

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.

Guinea Pig Model for Evaluating the Potential Public Health Risk of Swine and Avian Influenza Viruses

BackgroundThe influenza viruses circulating in animals sporadically transmit to humans and pose pandemic threats. Animal models to evaluate the potential public health risk potential of these viruses are needed.Methodology/Principal FindingsWe investigated the guinea pig as a mammalian model for the study of the replication and transmission characteristics of selected swine H1N1, H1N2, H3N2 and avian H9N2 influenza viruses, compared to those of pandemic (H1N1) 2009 and seasonal human H1N1, H3N2 influenza viruses. The swine and avian influenza viruses investigated were restricted to the respiratory system of guinea pigs and shed at high titers in nasal tracts without prior adaptation, similar to human strains. None of the swine and avian influenza viruses showed transmissibility among guinea pigs; in contrast, pandemic (H1N1) 2009 virus transmitted from infected guinea pigs to all animals and seasonal human influenza viruses could also horizontally transmit in guinea pigs. The analysis of the receptor distribution in the guinea pig respiratory tissues by lectin histochemistry indicated that both SAα2,3-Gal and SAα2,6-Gal receptors widely presented in the nasal tract and the trachea, while SAα2,3-Gal receptor was the main receptor in the lung.Conclusions/SignificanceWe propose that the guinea pig could serve as a useful mammalian model to evaluate the potential public health threat of swine and avian influenza viruses.

Pathogenicity of swine influenza viruses possessing an avian or swine-origin PB2 polymerase gene evaluated in mouse and pig models

PB2 627K is a determinant of influenza host range and contributes to the pathogenicity of human-, avian-, and mouse-adapted influenza viruses in the mouse model. Here we used mouse and pig models to analyze the contribution of a swine-origin and avian-origin PB2 carrying either 627K or 627E in the background of the classical swine H1N1 (A/Swine/Iowa/15/30; 1930) virus. The results showed PB2 627K is crucial for virulence in the mouse model, independent of whether PB2 is derived from an avian or swine influenza virus (SIV). In the pig model, PB2 627E decreases pathogenicity of the classical 1930 SIV when it contains the swine-origin PB2, but not when it possesses the avian-origin PB2. Our study suggests the pathogenicity of SIVs with different PB2 genes and mutation of codon 627 in mice does not correlate with the pathogenicity of the same SIVs in the natural host, the pig.

Origin of the 1918 pandemic H1N1 influenza A virus as studied by codon usage patterns and phylogenetic analysis

The pandemic of 1918 was caused by an H1N1 influenza A virus, which is a negative strand RNA virus; however, little is known about the nature of its direct ancestral strains. Here we applied a broad genetic and phylogenetic analysis of a wide range of influenza virus genes, in particular the PB1 gene, to gain information about the phylogenetic relatedness of the 1918 H1N1 virus. We compared the RNA genome of the 1918 strain to many other influenza strains of different origin by several means, including relative synonymous codon usage (RSCU), effective number of codons (ENC), and phylogenetic relationship. We found that the PB1 gene of the 1918 pandemic virus had ENC values similar to the H1N1 classical swine and human viruses, but different ENC values from avian as well as H2N2 and H3N2 human viruses. Also, according to the RSCU of the PB1 gene, the 1918 virus grouped with all human isolates and "classical" swine H1N1 viruses. The phylogenetic studies of all eight RNA gene segments of influenza A viruses may indicate that the 1918 pandemic strain originated from a H1N1 swine virus, which itself might be derived from a H1N1 avian precursor, which was separated from the bulk of other avian viruses in toto a long time ago. The high stability of the RSCU pattern of the PB1 gene indicated that the integrity of RNA structure is more important for influenza virus evolution than previously thought.

Genetic diversity of H9N2 influenza viruses from pigs in China: A potential threat to human health?

Pandemic strains of influenza A virus might arise by genetic reassortment between viruses from different hosts. Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of 10 (A–J) genotypes in pigs in China by analyzing the eight genes of 28 swine H9N2 viruses isolated in China from 1998 to 2007. Swine H9N2 viruses in genotype A and B were completely derived from Y280-like and Shanghai/F/98-like viruses, respectively, which indicated avian-to-pig interspecies transmission of H9N2 viruses did exist in China. The other eight genotype (C–J) viruses might be double-reassortant viruses, in which six genotype (E–J) viruses possessed 1–4 H5-like gene segments indicating they were reassortants of H9 and H5 viruses. In conclusion, genetic diversity of H9N2 influenza viruses from pigs in China provides further evidence that avian to pig interspecies transmission of H9N2 viruses did occur and might result in the generation of new reassortant viruses by genetic reassortment with swine H1N1, H1N2 and H3N2 influenza viruses, therefore, these swine H9N2 influenza viruses might be a potential threat to human health and continuing to carry out swine influenza virus surveillance in China is of great significance.

The 2009 Pandemic H1N1 and Triple-Reassortant Swine H1N1 Influenza Viruses Replicate Efficiently but Elicit an Attenuated Inflammatory Response in Polarized Human Bronchial Epithelial Cells

The pandemic H1N1 virus of 2009 (2009 H1N1) produced a spectrum of disease ranging from mild illness to severe illness and death. Respiratory symptoms were frequently associated with virus infection, with relatively high rate of gastrointestinal symptoms reported. To better understand 2009 H1N1 virus pathogenesis in humans, we studied virus and host responses following infection of two cell types: polarized bronchial and pharyngeal epithelial cells, which exhibit many features of the human airway epithelium, and colon epithelial cells to serve as a human intestinal cell model. Selected 2009 H1N1 viruses were compared to both seasonal H1N1 and triple-reassortant swine H1N1 influenza viruses that have circulated among North American pigs since before the 2009 pandemic. All H1N1 viruses replicated productively in airway cells; however, in contrast to seasonal H1N1 virus infection, infection with the 2009 H1N1 and triple-reassortant swine H1N1 viruses resulted in an attenuated inflammatory response, a weaker interferon response, and reduced cell death. Additionally, the H1N1 viruses of swine origin replicated less efficiently at the temperature of the human proximal airways (33°C). We also observed that the 2009 H1N1 viruses replicated to significantly higher titers than seasonal H1N1 virus in polarized colon epithelial cells. These studies reveal that in comparison to seasonal influenza virus, H1N1 viruses of swine origin poorly activate multiple aspects of the human innate response, which may contribute to the virulence of these viruses. In addition, their less efficient replication at human upper airway temperatures has implications for the understanding of pandemic H1N1 virus adaptation to humans.

Influenza A Virus Replication Induces Cell Cycle Arrest in G 0 /G 1 Phase

Many viruses interact with the host cell division cycle to favor their own growth. In this study, we examined the ability of influenza A virus to manipulate cell cycle progression. Our results show that influenza A virus A/WSN/33 (H1N1) replication results in G(0)/G(1)-phase accumulation of infected cells and that this accumulation is caused by the prevention of cell cycle entry from G(0)/G(1) phase into S phase. Consistent with the G(0)/G(1)-phase accumulation, the amount of hyperphosphorylated retinoblastoma protein, a necessary active form for cell cycle progression through late G(1) into S phase, decreased after infection with A/WSN/33 (H1N1) virus. In addition, other key molecules in the regulation of the cell cycle, such as p21, cyclin E, and cyclin D1, were also changed and showed a pattern of G(0)/G(1)-phase cell cycle arrest. It is interesting that increased viral protein expression and progeny virus production in cells synchronized in the G(0)/G(1) phase were observed compared to those in either unsynchronized cells or cells synchronized in the G(2)/M phase. G(0)/G(1)-phase cell cycle arrest is likely a common strategy, since the effect was also observed in other strains, such as H3N2, H9N2, PR8 H1N1, and pandemic swine H1N1 viruses. These findings, in all, suggest that influenza A virus may provide favorable conditions for viral protein accumulation and virus production by inducing a G(0)/G(1)-phase cell cycle arrest in infected cells.

Isolation and genetic analysis of a novel triple-reassortant H1N1 influenza virus from a pig in China

Influenza A viruses of subtype H1N1 have been reported widely in pigs in China, associated with clinical disease. These mainly include classical swine H1N1, avian-like H1N1, and human-like H1N1 viruses. In this study, we reported a novel triple-reassortant H1N1 virus (A/swine/Guangdong/1/2010) containing genes from the classical swine (NP, NS), human (PB1) and avian (HA, NA, M, PB2, PA) lineages, which was for the first time reported in China. Also, phylogenetic analysis further confirmed that five genes segments (NS, NP, PB2, PB1, PA) of the isolate were closely related to the novel reassortant H1N2 viruses isolated in China in 2006, while the other three (HA, NA, M) were closely related to avian-like H1N1 viruses in China. The isolation of triple-reassortant H1N1 influenza virus provides further evidence that pigs serve as emergence hosts or “mixing vessels”, and swine influenza virus (SIV) surveillance in China should be given a high priority.

Pandemic 2009 H1N1 vaccine protects against 1918 Spanish influenza virus

The 1918 influenza A virus caused the most devastating pandemic, killing approximately 50 million people worldwide. Immunization with 1918-like and classical swine H1N1 virus vaccines results in cross-protective antibodies against the 2009 H1N1 pandemic influenza, indicating antigenic similarities among these viruses. In this study, we demonstrate that vaccination with the 2009 pandemic H1N1 vaccine elicits 1918 virus cross-protective antibodies in mice and humans, and that vaccination or passive transfer of human-positive sera reduced morbidity and conferred full protection from lethal challenge with the 1918 virus in mice. The spread of the 2009 H1N1 influenza virus in the population worldwide, in addition to the large number of individuals already vaccinated, suggests that a large proportion of the population now have cross-protective antibodies against the 1918 virus, greatly alleviating concerns and fears regarding the accidental exposure/release of the 1918 virus from the laboratory and the use of the virus as a bioterrorist agent.

Swine Influenza A Vaccines, Pandemic (H1N1) 2009 Virus, and Cross-Reactivity

To the Editor: Since its first emergence in the human population in spring 2009 (1–3) infections with pandemic (H1N1) 2009 virus have been reported in pigs, turkeys, and some carnivore species (4,5). The pandemic (H1N1) 2009 virus can be experimentally transmitted between pigs (6). The reported transmissibility of the virus raises the question as to whether authorized swine influenza vaccine strains may be cross-reactive to pandemic (H1N1) 2009 virus. Kyriakis et al. (7) investigated the cross-reactivity of 66 pig serum samples from different infection and vaccination trials and reported cross-reactions between the avian-like H1N1 viruses circulating in the European pig population (avH1N1) and the classical swine H1N1 viruses (cH1N1) with pandemic (H1N1) 2009 virus by hemagglutination inhibition assay. To investigate this cross-reactivity in more detail, a neutralization test was applied in the study we report here. A serial dilution of serum samples was prepared (log4). All virus strains were adjusted to 100 fifty-percent tissue culture infectious doses. This working dilution of virus was mixed with serum dilutions and incubated 1 hour at 37°C. Madin-Darby bovine kidney monolayers were infected with the neutralization mixtures. After 48 hours of incubation, cells were fixed with acetone (4°C–8°C) and investigated by indirect immunofluorescent assay. Finally, the 50% neutralization titer was calculated. Hyperimmune serum samples were established by using a 4-fold vaccination of pigs with antigens of H1N1 vaccine strains (A/New Jersey/8/1976, A/sw/Netherlands/25/1980, A/sw/IDT/Re230/1992, A/sw/Haselunne/IDT2617/2003), and a strain of pandemic (H1N1) 2009 virus (A/Hamburg/7/2009) by using Freund adjuvant. Blood samples were taken 14 days after last immunization. A vaccine containing the pandemic (H1N1) 2009 virus was produced. Swine influenza vaccines available in central Europe and the newly produced vaccine containing pandemic (H1N1) 2009 virus (A/Hamburg/7/2009) were administered to pigs (2-fold vaccination with 1–2 mL of the vaccine 21–28 days apart intramuscularly). Blood was withdrawn 7 days after second administration. In addition, an experimental aerosol infection was conducted by using the parental strain of the most recent avH1N1 strain contained in a European swine influenza vaccine (A/sw/Haselunne/IDT2617/2003). Blood samples were taken 10 days after infection. The investigation of the hyperimmune serum samples detected neutralizing activity between the pandemic (H1N1) 2009 virus and European avH1N1 vaccine strains (A/sw/Netherlands/25/1980, A/sw/IDT/Re230/1992, A/sw/Haselunne/IDT2617/2003), as well as with the cH1N1 strain A/New Jersey/8/1976 (Fort Dix reassortant). The hyperimmune serum established against pandemic (H1N1) 2009 virus also showed cross-reactivity with European avH1N1 virus. The reactions against several strains of the pandemic virus were similar, reflecting high titers against pandemic (H1N1) 2009 virus but also cross-reactions with hyperimmune serum samples of all swine influenza A virus H1N1 vaccine strains (Appendix Table). The bivalent vaccines induced high titers of neutralizing antibodies against avH1N1 virus and human-like H3N2 virus (huH3N2). Only a low number of pigs reacted with H1N2 virus whereas the trivalent vaccine induced high neutralizing activity in serum samples of all vaccinated pigs. The vaccines induced neutralizing antibodies against pandemic (H1N1) 2009 virus. The titers were lower in comparison to those obtained for avH1N1 and not all pigs responded. The reactions were best for the vaccines containing mineral oil. Pigs vaccinated with the trivalent vaccine with carbomer adjuvant showed almost no antibodies against pandemic (H1N1) 2009 virus, although the vaccine strain reacted well in hyperimmunization tests. A vaccine batch of the trivalent vaccine was produced that contained mineral oil instead of carbomer. All pigs vaccinated with the trivalent vaccine with mineral oil had antibodies against the pandemic (H1N1) 2009 virus (data not shown). At the same time, efficacy trials with all authorized vaccines were conducted (8; T.W. Vahlenkamp, pers. comm.) in which all vaccines including the trivalent vaccine with carbomer adjuvant showed a comparable level of protection (limited period of viral shedding). Mineral oil adjuvants can induce severe distress in pig herds due to their limited safety. Despite cross-reactivity between avH1N1 and cH1N1 with pandemic (H1N1) 2009 virus, the highest degree of cross-neutralization was achieved by the vaccine containing pandemic (H1N1) virus strain. Proof of cross-reactivity was also reflected in the infection trial. Pigs infected with avH1N1 responded to avH1N1 as well as to pandemic (H1N1) 2009 virus. All results were additionally confirmed by hemagglutination inhibition assay (data not shown). Furthermore, 1,559 pig serum samples from 195 German pig herds collected from mid-June through mid-September 2009 were tested in routine diagnostics by hemagglutination inhibition assay. All reflected almost similar results for avH1N1 and the pandemic (H1N1) 2009 virus (seroprevalences for individual pigs were pandemic [H1N1] 2009 virus 52%, avH1N1 53%, huH1N2 28%, and huH3N2 52%; for pig herds pandemic [H1N1] 2009 virus 46%, avH1N1 46%, huH1N2 24%, and huH3N2 45%). These results suggest cross-reactivity between porcine H1N1 viruses and pandemic (H1N1) 2009 virus. Despite this cross-reactivity, a vaccine consisting of pandemic (H1N1) 2009 virus is superior in terms of efficacy in comparison with vaccines already authorized in Europe.

Prior infection of pigs with swine influenza viruses is a barrier to infection with avian influenza viruses

Although pigs are susceptible to avian influenza viruses (AIV) of different subtypes, the incidence of AIV infections in the field appears to be low. Swine H1N1, H3N2 and H1N2 influenza viruses (SIV) are enzootic worldwide and most pigs have antibodies to 1 or more SIV subtypes. This study aimed to examine whether infection–immunity to H1N1 or H3N2 SIV may (1) protect pigs against subsequent infections with AIV of various haemagglutinin and/or neuraminidase subtypes and/or (2) interfere with the serological diagnosis of AIV infection by haemagglutination inhibition (HI) or virus neutralization (VN) tests. Pigs were inoculated intranasally with an H1N1 or H3N2 SIV or left uninoculated. Four or 6 weeks later all pigs were challenged intranasally with 1 of 3 AIV subtypes (H4N6, H5N2 or H7N1). Fifteen out of 17 challenge control pigs shed the respective AIV for 4–6 days post-inoculation and 16 developed HI and VN antibodies. In contrast, 28 of the 29 SIV-immune pigs did not have detectable AIV shedding. Only 12 SIV-immune pigs developed HI antibodies to the AIV used for challenge and 14 had VN antibodies. Antibody titres to the AIV were low in both control and SIV-immune pigs. Our data show that prior infection of pigs with SIV is a barrier to infection with AIV of unrelated subtypes. Serological screening in regions where SIV is enzootic is only useful when the AIV strain for which the pigs need to be tested is known.

Prior infection with classical swine H1N1 influenza viruses is associated with protective immunity to the 2009 pandemic H1N1 virus.

Please cite this paper as: Kash et al. (2010) Prior infection with classical swine H1N1 influenza viruses is associated with protective immunity to the 2009 pandemic H1N1 virus. Influenza and Other Respiratory Viruses 4(3), 121–127. Background The 2009 H1N1 pandemic emerged even though seasonal H1N1 viruses have circulated for decades. Epidemio‐logical evidence suggested that the current seasonal vaccine did not offer significant protection from the novel pandemic, and that people over the age of 50 might were less susceptible to infection. Objectives In a mouse challenge study with the 2009 pandemic H1N1 virus, we evaluated protective immune responses elicited by prior infection with human and swine influenza A viruses. Results Mice infected with A/Mexico/4108/2009 (Mex09) showed significant weight loss and 40% mortality. Prior infection with a 1976 classical swine H1N1 virus resulted in complete protection from Mex09 challenge. Prior infection with either a 2009 or a 1940 seasonal H1N1 influenza virus provided partial protection and a >100‐fold reduction in viral lung titers at day 4 post‐infection. Conclusions These findings indicate that in experimental animals recently induced immunity to 1918‐derived H1N1 seasonal influenza viruses, and to a 1976 swine influenza virus, afford a degree of protection against the 2009 pandemic virus. Implications of these findings are discussed in the context of accumulating data suggesting partial protection of older persons during the 2009 pandemic.

Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.

Recombination analysis of full-length genomic sequences of novel influenza virus A/H1N1 in 2009 pandemic

Objective:To analyze the recombination of full-length genomic sequences of novel influenza virus A/H1N1 in 2009 pandemic.Methods:The full-length sequences of the novel A/H1N1 and reference sequences were downloaded from NCBI database.MEGA4.0 software was used to connect,align sequences,and analyze the similarity between the full-length sequences of the novel virus and each of the reference strains.Recombination was analyzed by Simplot software(version 3.5.1).Results:Simplot analysis indicated that the PB1 genes(polymerase B1,PB1)of the novel A/H1N1 viruses might evolve from human H3N2 virus(identity:93.7%);the PB2 genes(polymerase B2,PB2)and the PA genes(polymerase A,PA)might evolve from avian H5N1 viruses(identity:89.0%,89.9%,respectively);the HA genes(hemagglutinin,HA),the NP genes(nucleoprotein,NP)and the NS genes(non-structural protein,NS)showed high similarities with those of swine H1N1 viruses isolated in North America(identity:91.7%,93.1%,and 93.1%,respectively);and the NA genes(neuraminidase,NA)and the MP genes(matrix protein,MP)might evolve from European swine H1N1 viruses(identity:90.5%,95.5%,respectively).The full-length sequence of the novel A/H1N1 viruses had a highest similarities with swine H1N1 viruses isolated in North America(identity:83.9%).Conclusion:The novel influenza virus A/H1N1 is a recombinant virus evolving from human H3N2 viruses,swine H1N1 from North America,swine H1N1 from Europe,and swine H5N1 from Asia.

Attacking the flu: New prospects for the rational design of antivirals

Attacking the flu: New prospects for the rational design of antivirals

Detection of human novel influenza A (H1N1) viruses using multi-fluorescent real-time RT-PCR

The novel influenza A (H1N1) virus is now rapidly spreading across the world. Early detection is one of the most effective measures to prevent further transmission of the virus. 4 sets of proprietary primers and probes designed for detection of influenza A viruses (InfA), human and swine H1N1 viruses (SH1), the novel H1N1 viruses (NH1) and RNaseP gene (RP) respectively were pooled together in a single tube for a multi-fluorescent real-time RT-PCR assay. The detection limit was found to be one order more sensitive than that employing the WHO recommended protocol. The NH1 probe was negative for all control samples including human seasonal H1N1 virus, other subtypes of human influenza A viruses (H3, H5, H9), human influenza B virus and nasopharyngeal swabs from patients with noninfluenza respiratory diseases, indicating its high specificity, capable of discriminating the novel influenza A virus from the previously identified H1N1 viruses. For confirmation, the PCR amplified fragment of the hemagglutinin gene was sequenced which could provide enough information to identify the novel H1N1 virus as a distinct cluster among all viruses of subtype H1 through average distance clustering analysis. Although these assays should be useful in the current outbreak for rapid detection and discrimination of the novel H1N1 from swine H1N1 and other human seasonal H1N1 viruses, further design improvement is suggested to match possible sequence variations in the detected region along with the course of the epidemic.