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Long-term culture of the human lung adenocarcinoma cell line A549 promotes the differentiation of these cells toward an alveolar type II cell phenotype. Here, we evaluated the susceptibility of long-term cultured A549 cells to human influenza viruses. A549 cells were cultured continuously for 25 days (D25-A549) or 1 day (D1-A549) in Ham’s F12K medium. Six human influenza A viruses grew much faster in D25-A549 cells than in D1-A549 cells; however, two influenza B viruses replicated poorly in both cell types. Two avian influenza viruses replicated efficiently in both cell types, with similar titres. Expression levels of human virus receptors were higher in D25-A549 cells than in D1-A549 cells. D25-A549 cells thus more efficiently support the replication of human influenza A viruses compared with D1-A549 cells. Our data suggest that long-term cultured A549 cells will be useful for influenza A virus research.

Objectives Broadly reactive human monoclonal antibodies against the HA stem of influenza A virus are being developed as therapeutic agents as well as to understand the epitopes that are essential for a universal influenza virus vaccine. Methods We isolated and characterized two hetero-reactive human monoclonal antibodies from an H3N2 virus-infected human. Results These antibodies, which are predominantly bound to the HA stem of group 2 HAs, used IGHV3-66 and IGHV4-38-2 germline genes, respectively. They possessed in vitro neutralizing ability, and in vivo protective efficacy against lethal infection with H3N2 or H7N9 virus. Escape mutations revealed that one of the protective antibodies recognized the α-helix A of HA2, and the other recognized the C-terminal portion of the fusion peptide and the β-sheet that precedes the α-helix A of HA2. Conclusions Of many human protective monoclonal antibodies against the HA stem, two human protective monoclonal antibodies were isolated in this study that predominantly recognize epitopes on the HA stem of group 2 and use unique IGHV3-66 and IGHV4-38-2 germline genes.

Many broadly reactive human monoclonal antibodies against the hemagglutinin (HA) stem of influenza A virus have been developed for therapeutic applications. These antibodies typically inhibit viral entry steps, especially the HA conformational change that is required for membrane fusion. To better understand the mechanisms by which such antibodies inhibit viral replication, we established broadly reactive human anti-HA stem antibodies and determined the properties of these antibodies by examining their reactivity with 18 subtypes of HA, evaluating their in vivo protective efficacy, identifying their epitopes, and characterizing their inhibitory mechanisms. Among the eight human monoclonal antibodies we generated, which recognized at least 3 subtypes of the soluble HA antigens tested, clone S9-1-10/5-1 reacted with 18 subtypes of HA and protected mice from lethal infection with H1N1pdm09, H3N2, H5N1, and H7N9 viruses. This antibody recognized the HA2 helix A in the HA stem, and inhibited virus particle release from infected cells but did not block viral entry completely. These results show that broadly reactive human anti-HA stem antibodies can exhibit protective efficacy by inhibiting virus particle release. These findings expand our knowledge of the mechanisms by which broadly reactive stem-targeting antibodies inhibit viral replication and provide valuable information for universal vaccine development., Highlights • A broadly mouse-protective anti-HA stem antibody, S9-1-10/5-1, was isolated. • S9-1-10/5-1 mainly inhibited virus release rather than virus entry. • S9-1-10/5-1 tethers virions via crosslinking HA molecules between neighboring virions. Broadly reactive human monoclonal antibodies against the influenza HA stem have received attention because of their potential utility against multiple HA subtypes. Some of these antibodies inhibit virus entry and/or protect mice via antibody-dependent cellular cytotoxicity. Here, we identified a human monoclonal antibody that suppresses virus propagation in vitro and in vivo by primarily inhibiting virus particle release. This finding provides another inhibitory mechanism of action for the anti-HA stem antibodies, indicating that the anti-HA stem antibodies could be potent anti-virals due to their pluripotency.

Here we report the isolation of the influenza A/H1N1 2009 pandemic (A/H1N1pdm) and A/H3N2 viruses carrying an I38T mutation in the polymerase acidic protein-a mutation that confers reduced susceptibility to baloxavir marboxil-from patients before and after treatment with baloxavir marboxil in Japan. These variants showed replicative abilities and pathogenicity that is similar to those of wild-type isolates in hamsters; they also transmitted efficiently between ferrets by respiratory droplets.

Highly pathogenic avian H5 influenza viruses persist among poultry and wild birds throughout the world. They sometimes cause interspecies transmission between avian and mammalian hosts. H5 viruses possessing the HA of subclade 2.3.4.4, 2.3.2.1, 2.2.1, or 7.2 were detected between 2015 and 2018. To understand the neutralizing epitopes of H5-HA, we characterized 15 human monoclonal antibodies (mAbs) against the HA of H5 viruses, which were obtained from volunteers who received the H5N1 vaccine that contains a subclade 2.2.1 or 2.1.3.2 virus as an antigen. Twelve mAbs were specific for the HA of subclade 2.2.1, two mAbs were specific for the HA of subclade 2.1.3.2, and one mAb was specific for the HA of both. Of the 15 mAbs analyzed, nine, which were specific for the HA of subclade 2.2.1, and shared the VH and VL genes, possessed hemagglutination inhibition and neutralizing activities, whereas the others did not. A single amino acid substitution or insertion at positions 144&ndash, 147 in antigenic site A conferred resistance against these nine mAbs to the subclade 2.2.1 viruses. The amino acids at positions 144&ndash, 147 are highly conserved among subclade 2.2.1, but differ from those of other subclades. These results show that the neutralizing epitope including amino acids at positions 144&ndash, 147 is targeted by human antibodies, and plays a role in the antigenic difference between subclade 2.2.1 and other subclades.

Many cases of human infection with the H7N9 virus have been detected in China since 2013. H7N9 viruses are maintained in chickens and are transmitted to humans at live bird markets. During circulation in birds, H7N9 viruses have accumulated amino acid substitutions in their hemagglutinin (HA), which resulted in an antigenically change in the recent H7N9 viruses. Here, we characterized 46 mouse monoclonal antibodies against the HA of the prototype strain. 16 H7-HA-specific monoclonal antibodies (mAbs) possessed hemagglutination inhibition (HI) and neutralization activities by recognizing the major antigenic site A, four other H7-HA-specific clones also showed HI and neutralizing activities via recognition of the major antigenic sites A and D, seven mAbs that reacted with several HA subtypes and possibly recognized the HA stem partially protected mice from lethal infection with prototype H7N9 virus, and the remaining 19 mAbs had neither HI nor neutralization activity. All human H7N9 viruses tested showed a similar neutralization sensitivity to the first group of 16 mAbs, whereas human H7N9 viruses isolated in 2016‒2017 were not neutralized by a second group of 4 mAbs. These results suggest that amino acid substitutions at the epitope of the second mAb group appear to be involved in the antigenic drift of the H7N9 viruses. Further analysis is required to fully understand the antigenic change in H7N9 viruses.

Background Broadly protective human monoclonal antibodies that recognize the conserved epitopes in the HA of influenza A virus are being developed as therapeutic agents. Emergence of resistant viruses must always be considered when developing therapeutic agents against influenza. Objectives We examined human hetero-reactive mAbs against the HA stem of influenza A virus for the ease with which escape mutant viruses emerged. Study design We attempted to generate the mutant viruses escaped from the hetero-reactive anti-HA stem antibodies. We also evaluated their protective efficacy, binding affinity, and epitopes. Results We obtained several human monoclonal antibodies (mAbs) that react with the HA of different HA subtypes of influenza A virus belonging to group 1. Upon attempting to generate escape mutant viruses, we found that the ease with which such viruses emerged differed among the mAbs; viruses barely escaped from two of the mAbs (clones S9-3-37 and F20C77), whereas escape from the third mAb (clone F5B7) occurred readily. Comparisons of the mAbs revealed that the HA stem epitopes, in vitro neutralization potency, binding affinity to H1-HA, and protective efficacy against lethal challenge with H1N1pdm09 virus were all comparable. Conclusions These results demonstrate the importance of determining the ease with which escape mutant viruses emerge when evaluating anti-HA stem antibodies as antiviral agents during preclinical testing.

Several animal models are used to study influenza viruses. Intranasal inoculation of animals with a liquid inoculum is one of the main methods used to experimentally infect animals with influenza virus; however, this method does not reflect the natural infection with influenza virus by contact or aerosol route. Aerosol inhalation methods have been established with several influenza viruses for mouse and ferret models, but few studies have evaluated inoculation routes in a nonhuman primates (NHP) model. Here, we performed the experimental infection of NHPs with a highly pathogenic H5N1 influenza virus via the aerosol route and demonstrated that aerosol infection had no effect on clinical outcome, but caused broader infection throughout all of the lobes of the lung compared with a non-aerosolized approach. Aerosol infection therefore represents an option for inoculation of NHPs in future studies.

Since the 2017 Southern Hemisphere influenza season, the A(H1N1)pdm09-like virus recommended for use in the vaccine was changed because human, but not ferret, sera distinguish A(H1N1)pdm09 viruses isolated after 2013 from the previously circulating strains. An amino acid substitution, lysine to glutamine, at position 166 (H3 numbering) in the major antigenic site of HA was reported to be responsible for the antigenic drift. Here, we obtained two anti-A(H1N1)pdm09 HA monoclonal antibodies that failed to neutralize viruses isolated after 2013 from a vaccinated volunteer. Escape mutations were identified at position 129, 165, or 166 in the major antigenic site of HA. Competitive growth of the escape mutant viruses with the wild-type virus revealed that some escape mutants possessing an amino acid substitution other than K166Q showed superior growth to that of the wild-type virus. These results suggest that in addition to the K166Q mutation that occurred in epidemic strains, other HA mutations can confer resistance to antibodies that recognize the K166 area, leading to emergence of epidemic strains with such mutations.

Background Treatment of immunocompromised, influenza virus–infected patients with the viral neuraminidase inhibitor oseltamivir often leads to the emergence of drug-resistant variants. Combination therapy with compounds that target different steps in the viral life cycle may improve treatment outcomes and reduce the emergence of drug-resistant variants. Methods Here, we infected immunocompromised nude mice with an influenza A virus and treated them with neuraminidase (oseltamivir, laninamivir) or viral polymerase (favipiravir) inhibitors, or combinations thereof. Results Combination therapy for 28 days increased survival times compared with monotherapy, but the animals died after treatment was terminated. Mono- and combination therapies did not consistently reduce lung virus titers. Prolonged viral replication led to the emergence of neuraminidase inhibitor–resistant variants, although viruses remained sensitive to favipiravir. Overall, favipiravir provided greater benefit than neuraminidase inhibitors. Conclusions Collectively, our data demonstrate that combination therapy in immunocompromised hosts increases survival times, but does not suppress the emergence of neuraminidase inhibitor–resistant variants.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are enzootic in poultry populations in different parts of the world, and have caused numerous human infections in recent years, particularly in Egypt. However, no sustained human-to-human transmission of these viruses has yet been reported. We tested nine naturally occurring Egyptian H5N1 viruses (isolated in 2014–2015) in ferrets and found that three of them transmitted via respiratory droplets, causing a fatal infection in one of the exposed animals. All isolates were sensitive to neuraminidase inhibitors. However, these viruses were not transmitted via respiratory droplets in three additional transmission experiments in ferrets. Currently, we do not know if the efficiency of transmission is very low or if subtle differences in experimental parameters contributed to these inconsistent results. Nonetheless, our findings heighten concern regarding the pandemic potential of recent Egyptian H5N1 influenza viruses.

Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

Pigs are considered a mixing vessel for the generation of novel pandemic influenza A viruses through reassortment because of their susceptibility to both avian and human influenza viruses. However, experiments to understand reassortment in pigs in detail have been limited because experiments with regular-sized pigs are difficult to perform. Miniature pigs have been used as an experimental animal model, but they are still large and require relatively large cages for housing. The microminipig is one of the smallest miniature pigs used for experiments. Introduced in 2010, microminipigs weigh around 10 kg at an early stage of maturity (6 to 7 months old) and are easy to handle. To evaluate the microminipig as an animal model for influenza A virus infection, we compared the receptor distribution of 10-week-old male pigs (Yorkshire Large White) and microminipigs. We found that both animals have SAα2,3Gal and SAα2,6Gal in their respiratory tracts, with similar distributions of both receptor types. We further found that the sensitivity of microminipigs to influenza A viruses was the same as that of larger miniature pigs. Our findings indicate that the microminipig could serve as a novel model animal for influenza A virus infection. IMPORTANCE The microminipig is one of the smallest miniature pigs in the world and is used as an experimental animal model for life science research. In this study, we evaluated the microminipig as a novel animal model for influenza A virus infection. The distribution of influenza virus receptors in the respiratory tract of the microminipig was similar to that of the pig, and the sensitivity of microminipigs to influenza A viruses was the same as that of miniature pigs. Our findings suggest that microminipigs represent a novel animal model for influenza A virus infection.

The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. An Asp-to-Gly change at position 222 of the receptor-binding protein hemagglutinin (HA) correlates with more-severe infections in humans. The amino acid at position 222 of HA contributes to receptor-binding specificity with Asp (typically found in human influenza viruses) and Gly (typically found in avian and classic H1N1 swine influenza viruses), conferring binding to human- and avian-type receptors, respectively. Here, we asked whether binding to avian-type receptors enhances influenza virus pathogenicity. We tested two 2009 pandemic H1N1 viruses possessing HA-222G (isolated from severe cases) and two viruses that possessed HA-222D. In glycan arrays, viruses possessing HA-222D preferentially bound to human-type receptors, while those encoding HA-222G bound to both avian- and human-type receptors. This difference in receptor binding correlated with efficient infection of viruses possessing HA-222G, compared to those possessing HA-222D, in human lung tissue, including alveolar type II pneumocytes, which express avian-type receptors. In a nonhuman primate model, infection with one of the viruses possessing HA-222G caused lung damage more severe than did infection with a virus encoding HA-222D, although these pathological differences were not observed for the other virus pair with either HA-222G or HA-222D. These data demonstrate that the acquisition of avian-type receptor-binding specificity may result in more-efficient infection of human alveolar type II pneumocytes and thus more-severe lung damage. Collectively, these findings suggest a new mechanism by which influenza viruses may become more pathogenic in mammals, including humans.

Since its emergence, the 2009 pandemic H1N1 virus has spread rapidly throughout the world. Previously, we reported that most individuals born after 1920 do not have cross-reactive virus-neutralizing antibodies against pandemic (H1N1) 2009 virus, indicating that they were immunologically naïve to the pandemic virus prior to its emergence. This finding provided us with an excellent opportunity for a seroepidemiological investigation of the transmission mode of the pandemic virus in the community. To gain insight into its transmission within communities, we performed a serosurvey for pandemic virus infection with schoolchildren at an elementary school in Tokyo, Japan, and their parents. We observed a high prevalence of neutralizing antibodies to the pandemic virus in the children at this school, although the percentage of children positive for the neutralizing antibodies varied among classrooms. While a much lower prevalence was observed among parents, seropositivity of the parents correlated with that of their schoolchildren. Moreover, many adults appeared to have experienced asymptomatic infection with the pandemic virus. These data suggest that the pandemic virus was readily transmitted among schoolchildren in elementary schools and that it was also transmitted from schoolchildren to their parents.

Amino acids at positions 627 and 701 in the PB2 protein (PB2-627 and PB2-701, respectively) of avian influenza A viruses affect virus replication in some mammalian cells. Highly pathogenic H5N1 influenza viruses possessing mammalian-type PB2-627 were detected during the Qinghai Lake outbreak in 2005 and spread to Europe and Africa. Via a database search, we found a high rate of viral isolates from Ratitae , including ostrich, possessing mammalian-type PB2-627 or -701. Here, we report that H5N1 avian influenza viruses possessing mammalian-type amino acids in PB2-627 or -701 are selected during replication in ostrich cells in vitro and in vivo.

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

Highly pathogenic H5N1 influenza A viruses continue to circulate among avian species and cause sporadic cases of human infection. Therefore, the threat of a pandemic persists. However, the human cases of H5N1 infection have been limited mainly to individuals in close contact with infected poultry. These findings suggest that the H5N1 viruses need to acquire adaptive mutations to gain a replicative advantage in mammalian cells to break through the species barrier. Many amino acid mutations of the polymerase complex have been reported to enhance H5N1 virus growth in mammalian cells; however, the mechanism for H5N1 virus of adaptation to humans remains unclear. Here, we propose that the PA of an H5N1 influenza virus isolated from a human in Vietnam (A/Vietnam/UT36285/2010 [36285]) increased the ability of an avian H5N1 virus (A/chicken/Vietnam/TY31/2005 [Ck/TY31]) to grow in human lung epithelial A549 cells. The five PA amino acid substitutions V44I, V127A, C241Y, A343T, and I573V, which are rare in H5N1 viruses from human and avian sources, enhanced the growth capability of this virus in A549 cells. Moreover, these mutations increased the pathogenicity of the virus in mice, suggesting that they contribute to adaptation to mammalian hosts. Intriguingly, PA-241Y, which 36285 encodes, is conserved in more than 90% of human seasonal H1N1 viruses, suggesting that PA-241Y contributes to virus adaptation to human lung cells and mammalian hosts. IMPORTANCE Many amino acid substitutions in highly pathogenic H5N1 avian influenza viruses have been shown to contribute to adaptation to mammalian hosts. However, no naturally isolated H5N1 virus has caused extensive human-to-human transmission, suggesting that additional, as-yet unidentified amino acid mutations are needed for adaptation to humans. Here, we report that five amino acid substitutions in PA (V44I, V127A, C241Y, A343T, and I573V) contribute to the replicative efficiency of H5N1 viruses in human lung cells and to high virulence in mice. These results are helpful for assessing the pandemic risk of isolates and further our understanding of the mechanism of H5N1 virus adaptation to mammalian hosts.

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.

Vaccination is one of the most effective preventive measures to combat influenza. Prospectively, cell culture-based influenza vaccines play an important role for robust vaccine production in both normal settings and urgent situations, such as during the 2009 pandemic. African green monkey Vero cells are recommended by the World Health Organization as a safe substrate for influenza vaccine production for human use. However, the growth of influenza vaccine seed viruses is occasionally suboptimal in Vero cells, which places limitations on their usefulness for enhanced vaccine production. Here, we present a strategy for the development of vaccine seed viruses with enhanced growth in Vero cells by changing an amino acid residue in the stem region of the HA2 subunit of the hemagglutinin (HA) molecule. This mutation optimized the pH for HA-mediated membrane fusion in Vero cells and enhanced virus growth 100 to 1,000 times in the cell line, providing a promising strategy for cell culture-based influenza vaccines.

Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway.

Vietnam is one of the countries most affected by highly pathogenic H5N1 influenza A viruses. To evaluate the potential pathogenicity in mammals of H5N1 viruses isolated from humans in Vietnam, we determined the sequences of all eight genes of 22 human isolates collected between 2003 and 2008 and compared their virulence in mice. The isolates were classified into clade 1 and clade 2.3.4 and differed in pathogenicity for mice. Whilst lysine at position 627 of PB2 (PB2-627K) is a critical virulence determinant for clade 2.3.4 viruses, asparagine at position 701 of PB2 and other unknown virulence determinants appear to be involved in the high pathogenicity of clade 1 viruses, warranting further studies to determine the factors responsible for the high virulence of H5N1 viruses in mammals.

ContextVery little is known about the frequency of generation and transmissibility of influenza B viruses with reduced sensitivity to neuraminidase inhibitors. Furthermore, transmission of resistant virus, whether influenza A or B, has not been recognized to date.ObjectiveTo assess the prevalence and transmissibility of influenza B viruses with reduced sensitivity to neuraminidase inhibitors.Design, Setting, and PatientsInvestigation of the neuraminidase inhibitor sensitivity of influenza B isolates from 74 children before and after oseltamivir therapy and from 348 untreated patients with influenza (including 66 adults) seen at 4 community hospitals in Japan during the 2004-2005 influenza season. Four hundred twenty-two viruses from untreated patients and 74 samples from patients after oseltamivir therapy were analyzed.Main Outcome MeasureSialidase inhibition assay was used to test the drug sensitivities of influenza B viruses. The neuraminidase and hemagglutinin genes of viruses showing reduced sensitivity to neuraminidase inhibitors were sequenced to identify mutations that have the potential to confer reduced sensitivity to these drugs.ResultsIn 1 (1.4%) of the 74 children who had received oseltamivir, we identified a variant with reduced drug sensitivity possessing a Gly402Ser neuraminidase substitution. We also identified variants with reduced sensitivity carrying an Asp198Asn, Ile222Thr, or Ser250Gly mutation in 7 (1.7%) of the 422 viruses from untreated patients. Review of the clinical and viral genetic information available on these 7 patients indicated that 4 were likely infected in a community setting, while the remaining 3 were probably infected through contact with siblings shedding the mutant viruses.ConclusionsIn this population, influenza B viruses with reduced sensitivity to neuraminidase inhibitors do not arise as frequently as resistant influenza A viruses. However, they appear to be transmitted within communities and families, requiring continued close monitoring.

The pandemic threat posed by highly pathogenic H5N1 influenza A viruses has created an urgent need for vaccines to protect against H5 virus infection. Because pathogenic viruses grow poorly in chicken eggs and their virulence poses a biohazard to vaccine producers, avirulent viruses produced by reverse genetics have become the preferred basis for vaccine production. Here, we investigated two key characteristics of potential H5 vaccine candidates: the hemaggutinin (HA) cleavage site sequence and its modification to attenuate virulence and the choice of background virus to provide a high-growth rate. We produced recombinant (6:2 reassortant) viruses that possessed a series of modified avirulent-type HA and neuraminidase genes, both of which were derived from an H5N1 human isolate. The other genes of these recombinant viruses were derived from donor virus strains known to grow well in eggs: the human strain A/Puerto Rico/8/34 (PR8) or an avian strain. All of the recombinant viruses grew well in eggs, were avirulent in chicks, and protected animals against infection with a wild-type virus. However, one of the recombinant viruses with an avian virus background acquired a mutation in the HA cleavage site sequence that conferred virulence potential to this virus. Moreover, vaccine candidates with the avian virus background were more virulent than those with the human virus background. We conclude that 6:2 recombinant viruses with a PR8 background are more suitable than those with an avian virus background for vaccine development and that the HA cleavage site sequence must be modified to minimize the potential for a vaccine virus to convert to a virulent form.

Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals., Author Summary Influenza viruses that originate from avian species likely have to acquire adapting amino acid changes to replicate efficiently in mammals. Two amino acid changes in the polymerase PB2 protein—a glutamic acid to lysine change at position 627 or an aspartic acid to asparagine change at position 701—are known to allow influenza viruses of avian origin to replicate efficiently in mammals. Interestingly, the pandemic H1N1 viruses (which possess an avian-like PB2 gene) do not encode the ‘human-type’ amino acids PB2-627K and PB2-701N. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of PB2-627K and allows efficient replication of highly pathogenic H5N1 and pandemic H1N1 viruses in mammalian species. We also present the X-ray crystal structure of the C-terminal portion of a pandemic H1N1 PB2 protein. The basic amino acid at position 591 fills a distinctive cleft found in the PB2 proteins of H5N1 viruses. We also speculate on the biological significance of the altered surface of the H1N1 PB2 protein.