Pathogenicity and transmission in pigs of the novel A(H3N2)v influenza virus isolated from humans and characterization of swine H3N2 viruses isolated in 2010-2011

Swine influenza virus (SIV) H3N2 with triple reassorted internal genes (TRIG) has been enzootic in Unites States since 1998. Transmission of the 2009 pandemic H1N1 (pH1N1) virus to pigs in the United States was followed by reassortment with endemic SIV, resulting in reassorted viruses that include novel H3N2 genotypes (rH3N2p). Between July and December 2011, 12 cases of human infections with swine-lineage H3N2 viruses containing the pandemic matrix (pM) gene [A(H3N2)v] were detected. Whole-genome analysis of H3N2 viruses isolated from pigs from 2009 to 2011 sequenced in this study and other available H3N2 sequences showed six different rH3N2p genotypes present in the U.S. swine population since 2009. The presence of the pM gene was a common feature among all rH3N2p genotypes, but no specific genotype appeared to predominate in the swine population. We compared the pathogenic, transmission, genetic, and antigenic properties of a human A(H3N2)v isolate and two swine H3N2 isolates, H3N2-TRIG and rH3N2p. Our in vivo study detected no increased virulence in A(H3N2)v or rH3N2p viruses compared to endemic H3N2-TRIG virus. Antibodies to cluster IV H3N2-TRIG and rH3N2p viruses had reduced cross-reactivity to A(H3N2)v compared to other cluster IV H3N2-TRIG and rH3N2p viruses. Genetic analysis of the hemagglutinin gene indicated that although rH3N2p and A(H3N2)v are related to cluster IV of H3N2-TRIG, some recent rH3N2p isolates appeared to be forming a separate cluster along with the human isolates of A(H3N2)v. Continued monitoring of these H3N2 viruses is necessary to evaluate the evolution and potential loss of population immunity in swine and humans.     

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.     

Vaccination of pigs against swine influenza viruses by using an NS1-truncated modified live-virus vaccine

Swine influenza viruses (SIV) naturally infect pigs and can be transmitted to humans. In the pig, genetic reassortment to create novel influenza subtypes by mixing avian, human, and swine influenza viruses is possible. An SIV vaccine inducing cross-protective immunity between different subtypes and strains circulating in pigs is highly desirable. Previously, we have shown that an H3N2 SIV (A/swine/Texas/4199-2/98 [TX98]) containing a deleted NS1 gene expressing a truncated NS1 protein of 126 amino acids, NS1black triangle126, was attenuated in swine. In this study, 4-week-old pigs were vaccinated with the TX98 NS1black triangle126 modified live virus (MLV). Ten days after boosting, pigs were challenged with wild-type homologous H3N2 or heterosubtypic H1N1 SIV and sacrificed 5 days later. The MLV was highly attenuated and completely protected against challenge with the homologous virus. Vaccinated pigs challenged with the heterosubtypic H1N1 virus demonstrated macroscopic lung lesions similar to those of the unvaccinated H1N1 control pigs. Remarkably, vaccinated pigs challenged with the H1N1 SIV had significantly lower microscopic lung lesions and less virus shedding from the respiratory tract than did unvaccinated, H1N1-challenged pigs. All vaccinated pigs developed significant levels of hemagglutination inhibition and enzyme-linked immunosorbent assay titers in serum and mucosal immunoglobulin A antibodies against H3N2 SIV antigens. Vaccinated pigs were seronegative for NS1, indicating the potential use of the TX98 NS1black triangle126 MLV as a vaccine to differentiate infected from vaccinated animals.     

A contributing role for anti-neuraminidase antibodies on immunity to pandemic H1N1 2009 influenza A virus

Background

Exposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown.

Methods and Results

Antibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera.

Conclusions

The NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies contribute to but are not sufficient for cross-reactive immunity to pH1N1.     

Identification of reassortant pandemic H1N1 influenza virus in Korean pigs

Since the 2009 pandemic human H1N1 influenza A virus emerged in April 2009, novel reassortant strains have been identified throughout the world. This paper describes the detection and isolation of reassortant strains associated with human pandemic influenza H1N1 and swine influenza H1N2 (SIV) viruses in swine populations in South Korea. Two influenza H1N2 reassortants were detected, and subtyped by PCR. The strains were isolated using Madin- Darby canine kidney (MDCK) cells, and genetically characterized by phylogenetic analysis for genetic diversity. They consisted of human, avian, and swine virus genes that were originated from the 2009 pandemic H1N1 virus and a neuraminidase (NA) gene from H1N2 SIV previously isolated in North America. This identification of reassortment events in swine farms raises concern that reassortant strains may continuously circulate within swine populations, calling for the further study and surveillance of pandemic H1N1 among swine.     

Antigenic Characterization of H3N2 Influenza A Viruses from Ohio Agricultural Fairs

The demonstrated link between the emergence of H3N2 variant (H3N2v) influenza A viruses (IAVs) and swine exposure at agricultural fairs has raised concerns about the human health risk posed by IAV-infected swine. Understanding the antigenic profiles of IAVs circulating in pigs at agricultural fairs is critical to developing effective prevention and control strategies. Here, 68 H3N2 IAV isolates recovered from pigs at Ohio fairs (2009 to 2011) were antigenically characterized. These isolates were compared with other H3 IAVs recovered from commercial swine, wild birds, and canines, along with human seasonal and variant H3N2 IAVs. Antigenic cartography demonstrated that H3N2 IAV isolates from Ohio fairs could be divided into two antigenic groups: (i) the 2009 fair isolates and (ii) the 2010 and 2011 fair isolates. These same two antigenic clusters have also been observed in commercial swine populations in recent years. Human H3N2v isolates from 2010 and 2011 are antigenically clustered with swine-origin IAVs from the same time period. The isolates recovered from pigs at fairs did not cross-react with ferret antisera produced against the human seasonal H3N2 IAVs circulating during the past decade, raising the question of the degree of immunity that the human population has to swine-origin H3N2 IAVs. Our results demonstrate that H3N2 IAVs infecting pigs at fairs and H3N2v isolates were antigenically similar to the IAVs circulating in commercial swine, demonstrating that exhibition swine can function as a bridge between commercial swine and the human population.     

Genetic characterization of swine influenza viruses isolated in Japan between 2009 and 2012

Eleven swine influenza viruses (SIVs) isolated from pigs in Japanese institutions between 2009 and 2012 were genetically characterized. Seven H1N1 were shown to have originated from A(H1N1)pdm09 viruses. Two H1N2 viruses contained H1 and N2 genes of Japanese H1N2 SIV origin together with internal genes of A(H1N1)pdm09 viruses. Two H3N2 viruses isolated during animal quarantine were identified as triple reassortant H3N2 viruses maintained among pigs in North America. This study shows that A(H1N1)pdm09 viruses and their reassortant strains are already present in domestic pigs in Japan and that novel SIVs are possibly being imported from abroad.     

Reassortant swine influenza viruses isolated in Japan contain genes from pandemic A(H1N1) 2009

In 2013, three reassortant swine influenza viruses (SIVs)—two H1N2 and one H3N2—were isolated from symptomatic pigs in Japan; each contained genes from the pandemic A(H1N1) 2009 virus and endemic SIVs. Phylogenetic analysis revealed that the two H1N2 viruses, A/swine/Gunma/1/2013 and A/swine/Ibaraki/1/2013, were reassortants that contain genes from the following three distinct lineages: (i) H1 and nucleoprotein (NP) genes derived from a classical swine H1 HA lineage uniquely circulating among Japanese SIVs; (ii) neuraminidase (NA) genes from human‐like H1N2 swine viruses; and (iii) other genes from pandemic A(H1N1) 2009 viruses. The H3N2 virus, A/swine/Miyazaki/2/2013, comprised genes from two sources: (i) hemagglutinin (HA) and NA genes derived from human and human‐like H3N2 swine viruses and (ii) other genes from pandemic A(H1N1) 2009 viruses. Phylogenetic analysis also indicated that each of the reassortants may have arisen independently in Japanese pigs. A/swine/Miyazaki/2/2013 were found to have strong antigenic reactivities with antisera generated for some seasonal human‐lineage viruses isolated during or before 2003, whereas A/swine/Miyazaki/2/2013 reactivities with antisera against viruses isolated after 2004 were clearly weaker. In addition, antisera against some strains of seasonal human‐lineage H1 viruses did not react with either A/swine/Gunma/1/2013 or A/swine/Ibaraki/1/2013. These findings indicate that emergence and spread of these reassortant SIVs is a potential public health risk.     

Enhanced mammalian transmissibility of seasonal influenza A/H1N1 viruses encoding an oseltamivir-resistant neuraminidase

Between 2007 and 2009, oseltamivir resistance developed among seasonal influenza A/H1N1 (sH1N1) virus isolates at an exponential rate, without a corresponding increase in oseltamivir usage. We hypothesized that the oseltamivir-resistant neuraminidase (NA), in addition to being relatively insusceptible to the antiviral effect of oseltamivir, might confer an additional fitness advantage on these viruses by enhancing their transmission efficiency among humans. Here we demonstrate that an oseltamivir-resistant clinical isolate, an A/Brisbane/59/2007(H1N1)-like virus isolated in New York State in 2008, transmits more efficiently among guinea pigs than does a highly similar, contemporaneous oseltamivir-sensitive isolate. With reverse genetics reassortants and point mutants of the two clinical isolates, we further show that expression of the oseltamivir-resistant NA in the context of viral proteins from the oseltamivir-sensitive virus (a 7:1 reassortant) is sufficient to enhance transmissibility. In the guinea pig model, the NA is the critical determinant of transmission efficiency between oseltamivir-sensitive and -resistant Brisbane/59-like sH1N1 viruses, independent of concurrent drift mutations that occurred in other gene products. Our data suggest that the oseltamivir-resistant NA (specifically, one or both of the companion mutations, H275Y and D354G) may have allowed resistant Brisbane/59-like viruses to outtransmit sensitive isolates. These data provide in vivo evidence of an evolutionary mechanism that would explain the rapidity with which oseltamivir resistance achieved fixation among sH1N1 isolates in the human reservoir.     

Characterization of low-pathogenicity H5N1 avian influenza viruses from North America

Wild-bird surveillance in North America for avian influenza (AI) viruses with a goal of early identification of the Asian H5N1 highly pathogenic AI virus has identified at least six low-pathogenicity H5N1 AI viruses between 2004 and 2006. The hemagglutinin (HA) and neuraminidase (NA) genes from all 6 H5N1 viruses and an additional 38 North American wild-bird-origin H5 subtype and 28 N1 subtype viruses were sequenced and compared with sequences available in GenBank by phylogenetic analysis. Both HA and NA were phylogenetically distinct from those for viruses from outside of North America and from those for viruses recovered from mammals. Four of the H5N1 AI viruses were characterized as low pathogenicity by standard in vivo pathotyping tests. One of the H5N1 viruses, A/MuteSwan/MI/451072-2/06, was shown to replicate to low titers in chickens, turkeys, and ducks. However, transmission of A/MuteSwan/MI/451072-2/06 was more efficient among ducks than among chickens or turkeys based on virus shed. The 50% chicken infectious dose for A/MuteSwan/MI/451072-2/06 and three other wild-waterfowl-origin H5 viruses were also determined and were between 10(5.3) and 10(7.5) 50% egg infective doses. Finally, seven H5 viruses representing different phylogenetic clades were evaluated for their antigenic relatedness by hemagglutination inhibition assay, showing that the antigenic relatedness was largely associated with geographic origin. Overall, the data support the conclusion that North American H5 wild-bird-origin AI viruses are low-pathogenicity wild-bird-adapted viruses and are antigenically and genetically distinct from the highly pathogenic Asian H5N1 virus lineage.     

Genetic and phylogenetic analyses of influenza A H1N1pdm virus in Buenos Aires, Argentina

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.     

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.     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they 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 reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.     

Close relationship between the 2009 H1N1 virus and South Dakota AIV strains

Although previous publications suggest the 2009 pandemic influenza A (H1N1) virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the 2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the 2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1 virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.     

Genetic and antigenic characterization of swine H1N2 influenza viruses isolated from Korean pigs

H1N2 influenza viruses are circulating in pigs worldwide and cause considerable economic losses to the pig industry. We genetically analyzed the genes of our isolates from Korean pigs, and compared the antigenicity of our isolates with swine H1N2 viruses isolated from pigs in the U.S.A. In addition, we serologically surveyed the infection rate of swine H1N2 viruses in pigs. We found that H1N2 isolates from Korean pigs are genetically more related to swine H1N2 viruses isolated from pigs in the U.S.A. than those in European countries. When antigenicity was compared, our isolates were weakly reacted to antibodies against swine H1N2 viruses isolated from pigs in the U.S.A. The serological surveillance using sera from pigs in Korea showed that about 46% was positive for H1N2 viruses. Our results suggest that swine H1N2 viruses are widespread in Korean pigs, and the development of a vaccine against H1N2 viruses may help to control their infection in pigs.     

Tissue tropism of swine influenza viruses and reassortants in ex vivo cultures of the human respiratory tract and conjunctiva

The 2009 pandemic influenza H1N1 (H1N1pdm) virus was generated by reassortment of swine influenza viruses of different lineages. This was the first influenza pandemic to emerge in over 4 decades and the first to occur after the realization that influenza pandemics arise from influenza viruses of animals. In order to understand the biological determinants of pandemic emergence, it is relevant to compare the tropism of different lineages of swine influenza viruses and reassortants derived from them with that of 2009 pandemic H1N1 (H1N1pdm) and seasonal influenza H1N1 viruses in ex vivo cultures of the human nasopharynx, bronchus, alveoli, and conjunctiva. We hypothesized that virus which can transmit efficiently between humans replicated well in the human upper airways. As previously reported, H1N1pdm and seasonal H1N1 viruses replicated efficiently in the nasopharyngeal, bronchial, and alveolar epithelium. In contrast, representative viruses from the classical swine (CS) (H1N1) lineage could not infect human respiratory epithelium; Eurasian avian-like swine (EA) (H1N1) viruses only infected alveolar epithelium and North American triple-reassortant (TRIG) viruses only infected the bronchial epithelium albeit inefficiently. Interestingly, a naturally occurring triple-reassortant swine virus, A/SW/HK/915/04 (H1N2), with a matrix gene segment of EA swine derivation (i.e., differing from H1N1pdm only in lacking a neuraminidase [NA] gene of EA derivation) readily infected and replicated in human nasopharyngeal and bronchial epithelia but not in the lung. A recombinant sw915 with the NA from H1N1pdm retained its tropism for the bronchus and acquired additional replication competence for alveolar epithelium. In contrast to H1N1pdm, none of the swine viruses tested nor seasonal H1N1 had tropism in human conjunctiva. Recombinant viruses generated by swapping the surface proteins (hemagglutinin and NA) of H1N1pdm and seasonal H1N1 virus demonstrated that these two gene segments together are key determinants of conjunctival tropism. Overall, these findings suggest that ex vivo cultures of the human respiratory tract provide a useful biological model for assessing the human health risk of swine influenza viruses.     

Molecular characterization of avian-like H1N1 swine influenza a viruses isolated in Eastern China, 2011

Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2. European avian-like H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3′ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.     

Characterization of Oseltamivir-Resistant 2009 H1N1 Pandemic Influenza A Viruses

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.     

Impact of amino acid mutations in PB2, PB1-F2, and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses

Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.