Compatibility of H9N2 avian influenza surface genes and 2009 pandemic H1N1 internal genes for transmission in the ferret model

In 2009, a novel H1N1 influenza (pH1N1) virus caused the first influenza pandemic in 40 y. The virus was identified as a triple reassortant between avian, swine, and human influenza viruses, highlighting the importance of reassortment in the generation of viruses with pandemic potential. Previously, we showed that a reassortant virus composed of wild-type avian H9N2 surface genes in a seasonal human H3N2 backbone could gain efficient respiratory droplet transmission in the ferret model. Here we determine the ability of the H9N2 surface genes in the context of the internal genes of a pH1N1 virus to efficiently transmit via respiratory droplets in ferrets. We generated reassorted viruses carrying the HA gene alone or in combination with the NA gene of a prototypical H9N2 virus in the background of a pH1N1 virus. Four reassortant viruses were generated, with three of them showing efficient respiratory droplet transmission. Differences in replication efficiency were observed for these viruses; however, the results clearly indicate that H9N2 avian influenza viruses and pH1N1 viruses, both of which have occasionally infected pigs, have the potential to reassort and generate novel viruses with respiratory transmission potential in mammals.     

Minimal molecular constraints for respiratory droplet transmission of an avian–human H9N2 influenza A virus

Pandemic influenza requires interspecies transmission of an influenza virus with a novel hemagglutinin (HA) subtytpe that can adapt to its new host through either reassortment or point mutations and transmit by aerosolized respiratory droplets. Two previous pandemics of 1957 and 1968 resulted from the reassortment of low pathogenic avian viruses and human subtypes of that period; however, conditions leading to a pandemic virus are still poorly understood. Given the endemic situation of avian H9N2 influenza with human-like receptor specificity in Eurasia and its occasional transmission to humans and pigs, we wanted to determine whether an avian–human H9N2 reassortant could gain respiratory transmission in a mammalian animal model, the ferret. Here we show that following adaptation in the ferret, a reassortant virus carrying the surface proteins of an avian H9N2 in a human H3N2 backbone can transmit efficiently via respiratory droplets, creating a clinical infection similar to human influenza infections. Minimal changes at the protein level were found in this virus capable of respiratory droplet transmission. A reassortant virus expressing only the HA and neuraminidase (NA) of the ferret-adapted virus was able to account for the transmissibility, suggesting that currently circulating avian H9N2 viruses require little adaptation in mammals following acquisition of all human virus internal genes through reassortment. Hemagglutinin inhibition (HI) analysis showed changes in the antigenic profile of the virus, which carries profound implications for vaccine seed stock preparation against avian H9N2 influenza. This report illustrates that aerosolized respiratory transmission is not exclusive to current human H1, H2, and H3 influenza subtypes.     

Diversity of influenza viruses in swine and the emergence of a novel human pandemic influenza A (H1N1)

Abstract The novel H1N1 influenza virus that emerged in humans in Mexico in early 2009 and transmitted efficiently in the human population with global spread has been declared a pandemic strain. Here we review influenza infections in swine since 1918 and the introduction of different avian and human influenza virus genes into swine influenza viruses of North America and Eurasia. These introductions often result in viruses of increased fitness for pigs that occasionally transmit to humans. The novel virus affecting humans is derived from a North American swine influenza virus that has acquired two gene segments [Neuraminidase (NA) and Matrix (M)] from the European swine lineages. This reassortant appears to have increased fitness in humans. The potential for increased virulence in humans and of further reassortment between the novel H1N1 influenza virus and oseltamivir resistant seasonal H1N1 or with highly pathogenic H5N1 influenza stresses the need for urgent pandemic planning.     

Hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin-neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.     

High genetic compatibility and increased pathogenicity of reassortants derived from avian H9N2 and pandemic H1N1/2009 influenza viruses

H9N2 influenza viruses have been circulating worldwide in multiple avian species and repeatedly infecting mammals, including pigs and humans, posing a significant threat to public health. The coexistence of H9N2 and pandemic influenza H1N1/2009 viruses in pigs and humans provides an opportunity for these viruses to reassort. To evaluate the potential public risk of the reassortant viruses derived from these viruses, we used reverse genetics to generate 127 H9 reassortants derived from an avian H9N2 and a pandemic H1N1 virus, and evaluated their compatibility, replication ability, and virulence in mice. These hybrid viruses showed high genetic compatibility and more than half replicated to a high titer in vitro. In vivo studies of 73 of 127 reassortants revealed that all viruses were able to infect mice without prior adaptation and 8 reassortants exhibited higher pathogenicity than both parental viruses. All reassortants with higher virulence than parental viruses contained the PA gene from the 2009 pandemic virus, revealing the important role of the PA gene from the H1N1/2009 virus in generating a reassortant virus with high public health risk. Analyses of the polymerase activity of the 16 ribonucleoprotein combinations in vitro suggested that the PA of H1N1/2009 origin also enhanced polymerase activity. Our results indicate that some avian H9-pandemic reassortants could emerge with a potentially higher threat for humans and also highlight the importance of monitoring the H9-pandemic reassortant viruses that may arise, especially those that possess the PA gene of H1N1/2009 origin.     

Influenza pandemic--current crisis

In most cases, influenza is not fatal, even without treatment. Moreover, vaccination and antivirals have reduced influenza-related mortality in recent years. However, the direct transmission of avian influenza viruses to humans with lethal outcomes in Hong Kong in 1997 was a potent reminder of the devastating potential of the disease. Currently, H5N1 avian influenza viruses are circulating in many Asian countries, and the human death toll continues to rise as the virus spreads to European countries as well. Since the beginning of the outbreak in Asia, more than 120 cases have been confirmed and the mortality rate has been no less than 50%. Current vaccines for H3N2 and H1N1 viruses, of course, have no effect on infection by H5N1 viruses. In addition, H5N1 viruses that are resistant to the antiviral drugs amantadine and oseltamivir have emerged. Fortunately, a virus that is capable of efficient transmission among humans has not emerged. However, it is not a matter of if, but when, such a virus will appear. Here, we review the current situation of avian influenza and pandemic preparedness.     

Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus

The mechanisms responsible for the virulence of the highly pathogenic avian influenza (HPAI) and of the 1918 pandemic influenza virus in humans remain poorly understood. To identify crucial components of the early host response during these infections by using both conventional and functional genomics tools, we studied 34 cynomolgus macaques (Macaca fascicularis) to compare a 2004 human H5N1 Vietnam isolate with 2 reassortant viruses possessing the 1918 hemagglutinin (HA) and neuraminidase (NA) surface proteins, known conveyors of virulence. One of the reassortants also contained the 1918 nonstructural (NS1) protein, an inhibitor of the host interferon response. Among these viruses, HPAI H5N1 was the most virulent. Within 24 h, the H5N1 virus produced severe bronchiolar and alveolar lesions. Notably, the H5N1 virus targeted type II pneumocytes throughout the 7-day infection, and induced the most dramatic and sustained expression of type I interferons and inflammatory and innate immune genes, as measured by genomic and protein assays. The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection. While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.     

Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates

In 1997, 18 cases of influenza in Hong Kong (bird flu) caused by a novel H5N1 (chicken) virus resulted in the deaths of six individuals and once again raised the specter of a potentially devastating influenza pandemic. Slaughter of the poultry in the live bird markets removed the source of infection and no further human cases of H5N1 infection have occurred. In March 1999, however, a new pandemic threat appeared when influenza A H9N2 viruses infected two children in Hong Kong. These two virus isolates are similar to an H9N2 virus isolated from a quail in Hong Kong in late 1997. Although differing in their surface hemagglutinin and neuraminidase components, a notable feature of these H9N2 viruses is that the six genes encoding the internal components of the virus are similar to those of the 1997 H5N1 human and avian isolates. This common feature emphasizes the apparent propensity of avian viruses with this genetic complement to infect humans and highlights the potential for the emergence of a novel human pathogen.     

Human and avian influenza viruses target different cell types in cultures of human airway epithelium

The recent human infections caused by H5N1, H9N2, and H7N7 avian influenza viruses highlighted the continuous threat of new pathogenic influenza viruses emerging from a natural reservoir in birds. It is generally believed that replication of avian influenza viruses in humans is restricted by a poor fit of these viruses to cellular receptors and extracellular inhibitors in the human respiratory tract. However, detailed mechanisms of this restriction remain obscure. Here, using cultures of differentiated human airway epithelial cells, we demonstrated that influenza viruses enter the airway epithelium through specific target cells and that there were striking differences in this respect between human and avian viruses. During the course of a single-cycle infection, human viruses preferentially infected nonciliated cells, whereas avian viruses as well as the egg-adapted human virus variant with an avian virus-like receptor specificity mainly infected ciliated cells. This pattern correlated with the predominant localization of receptors for human viruses (2-6-linked sialic acids) on nonciliated cells and of receptors for avian viruses (2-3-linked sialic acids) on ciliated cells. These findings suggest that although avian influenza viruses can infect human airway epithelium, their replication may be limited by a nonoptimal cellular tropism. Our data throw light on the mechanisms of generation of pandemic viruses from their avian progenitors and open avenues for cell level-oriented studies on the replication and pathogenicity of influenza virus in humans.     

Avian influenza virus infections in humans

Seroepidemiologic and virologic studies since 1889 suggested that human influenza pandemics were caused by H1, H2, and H3 subtypes of influenza A viruses. If not for the 1997 avian A/H5N1 outbreak in Hong Kong of China, subtype H2 is the likely candidate for the next pandemic. However, unlike previous poultry outbreaks of highly pathogenic avian influenza due to H5 that were controlled by depopulation with or without vaccination, the presently circulating A/H5N1 genotype Z virus has since been spreading from Southern China to other parts of the world. Migratory birds and, less likely, bird trafficking are believed to be globalizing the avian influenza A/H5N1 epidemic in poultry. More than 200 human cases of avian influenza virus infection due to A/H5, A/H7, and A/H9 subtypes mainly as a result of poultry-to-human transmission have been reported with a > 50% case fatality rate for A/H5N1 infections. A mutant or reassortant virus capable of efficient human-to-human transmission could trigger another influenza pandemic. The recent isolation of this virus in extrapulmonary sites of human diseases suggests that the high fatality of this infection may be more than just the result of a cytokine storm triggered by the pulmonary disease. The emergence of resistance to adamantanes (amantadine and rimantadine) and recently oseltamivir while H5N1 vaccines are still at the developmental stage of phase I clinical trial are causes for grave concern. Moreover, the to-be pandemic strain may have little cross immunogenicity to the presently tested vaccine strain. The relative importance and usefulness of airborne, droplet, or contact precautions in infection control are still uncertain. Laboratory-acquired avian influenza H7N7 has been reported, and the laboratory strains of human influenza H2N2 could also be the cause of another pandemic. The control of this impending disaster requires more research in addition to national and international preparedness at various levels. The epidemiology, virology, clinical features, laboratory diagnosis, management, and hospital infection control measures are reviewed from a clinical perspective.     

Development of a new candidate H5N1 avian influenza virus for pre‐pandemic vaccine production

Background Highly pathogenic H5N1 avian influenza viruses currently circulating in birds have caused hundreds of human infections, and pose a significant pandemic threat. Vaccines are a major component of the public health preparedness for this likely event. The rapid evolution of H5N1 viruses has resulted in the emergence of multiple clades with distinct antigenic characteristics that require clade‐specific vaccines. A variant H5N1 virus termed clade 2.3.4 emerged in 2005 and has caused multiple fatal infections. Vaccine candidates that match the antigenic properties of variant viruses are necessary because inactivated influenza vaccines elicit strain‐specific protection. Objective To address the need for a suitable seed for manufacturing a clade 2.3.4 vaccine, we developed a new H5N1 pre‐pandemic candidate vaccine by reverse genetics and evaluated its safety and replication in vitro and in vivo. Methods A reassortant virus termed, Anhui/PR8, was produced by reverse genetics in compliance with WHO pandemic vaccine development guidelines and contains six genes from A/Puerto Rico/8/34 as well as the neuraminidase and hemagglutinin (HA) genomic segments from the A/Anhui/01/2005 virus. The multi‐basic cleavage site of HA was removed to reduce virulence. Results The reassortant Anhui/PR8 grows well in eggs and is avirulent to chicken and ferrets but retains the antigenicity of the parental A/Anhui/01/2005 virus. Conclusion These results indicate that the Anhui/PR8 reassortant lost a major virulent determinant and it is suitable for its use in vaccine manufacturing and as a reference vaccine virus against the H5N1 clade 2.3.4 viruses circulating in eastern China, Vietnam, Thailand, and Laos.     

Adaptive strategies of the influenza virus polymerase for replication in humans

Transmission of influenza viruses into the human population requires surmounting barriers to cross-species infection. Changes in the influenza polymerase overcome one such barrier. Viruses isolated from birds generally contain polymerases with the avian-signature glutamic acid at amino acid 627 in the PB2 subunit. These polymerases display restricted activity in human cells. An adaptive change in this residue from glutamic acid to the human-signature lysine confers high levels of polymerase activity in human cells. This mutation permits escape from a species-specific restriction factor that targets polymerases from avian viruses. A 2009 swine-origin H1N1 influenza A virus recently established a pandemic infection in humans, even though the virus encodes a PB2 with the restrictive glutamic acid at amino acid 627. We show here that the 2009 H1N1 virus has acquired second-site suppressor mutations in its PB2 polymerase subunit that convey enhanced polymerase activity in human cells. Introduction of this polymorphism into the PB2 subunit of a primary avian isolate also increased polymerase activity and viral replication in human and porcine cells. An alternate adaptive strategy has also been identified, whereby introduction of a human PA subunit into an avian polymerase overcomes restriction in human cells. These data reveal a strategy used by the 2009 H1N1 influenza A virus and identify other pathways by which avian and swine-origin viruses may evolve to enhance replication, and potentially pathogenesis, in humans.     

H5N1 influenza: a protean pandemic threat

Infection with avian influenza A virus of the H5N1 subtype (isolates A/HK/212/03 and A/HK/213/03) was fatal to one of two members of a family in southern China in 2003. This incident was preceded by lethal outbreaks of H5N1 influenza in waterfowl, which are the natural hosts of these viruses and, therefore, normally have asymptomatic infection. The hemagglutinin genes of the A/HK/212/03-like viruses isolated from humans and waterfowl share the lineage of the H5N1 viruses that caused the first known cases of human disease in Hong Kong in 1997, but their internal protein genes originated elsewhere. The hemagglutinin of the recent human isolates has undergone significant antigenic drift. Like the 1997 human H5N1 isolates, the 2003 human H5N1 isolates induced the overproduction of proinflammatory cytokines by primary human macrophages in vitro, whereas the precursor H5N1 viruses and other H5N1 reassortants isolated in 2001 did not. The acquisition by the viruses of characteristics that enhance virulence in humans and waterfowl and their potential for wider distribution by infected migrating birds are causes for renewed pandemic concern.     

Preparation of genetically engineered A/H5N1 and A/H7N1 pandemic vaccine viruses by reverse genetics in a mixture of Vero and chicken embryo cells

Background In case of influenza pandemic, a robust, easy and clean technique to prepare reassortants would be necessary. Objectives Using reverse genetics, we prepared two vaccine reassortants (A/H5N1 × PR8 and A/H7N1 × PR8) exhibiting the envelope glycoproteins from non‐pathogenic avian viruses, A/Turkey/Wisconsin/68 (A/H5N9) and A/Rhea/New Caledonia/39482/93 (A/H7N1) and the internal proteins of the attenuated human virus A/Puerto Rico/8/34 (H1N1). Methods The transfection was accomplished using a mixture of Vero and chicken embryo cells both of which are currently being used for vaccine manufacturing. Results This process was reproducible, resulting in consistent recovery of influenza viruses in 6 days. Because it is mainly the A/H5N1 strain that has recently crossed the human barrier, it is the A/PR8 × A/H5N1 reassortant (RG5) that was further amplified, either in embryonated hen eggs or Vero cells, to produce vaccine pre‐master seed stocks that met quality control specifications. Safety testing in chickens and ferrets was performed to assess the non‐virulence of the reassortant, and finally analysis using chicken and ferret sera immunized with the RG5 virus showed that the vaccine candidate elicited an antibody response cross‐reactive with the Hong Kong 1997 and 2003 H5N1 strains but not the Vietnam/2004 viruses. Conclusions The seeds obtained could be used as part of a pandemic vaccine strain ‘library’ available in case of propagation in humans of a new highly pathogenic avian strain.     

Identification of H2N3 influenza A viruses from swine in the United States

Although viruses of each of the 16 influenza A HA subtypes are potential human pathogens, only viruses of the H1, H2, and H3 subtype are known to have been successfully established in humans. H2 influenza viruses have been absent from human circulation since 1968, and as such they pose a substantial human pandemic risk. In this report, we isolate and characterize genetically similar avian/swine virus reassortant H2N3 influenza A viruses isolated from diseased swine from two farms in the United States. These viruses contained leucine at position 226 of the H2 protein, which has been associated with increased binding affinity to the mammalian α2,6Gal-linked sialic acid virus receptor. Correspondingly, the H2N3 viruses were able to cause disease in experimentally infected swine and mice without prior adaptation. In addition, the swine H2N3 virus was infectious and highly transmissible in swine and ferrets. Taken together, these findings suggest that the H2N3 virus has undergone some adaptation to the mammalian host and that their spread should be very closely monitored.     

From the Cover: Dating the emergence of pandemic influenza viruses

Pandemic influenza viruses cause significant mortality in humans. In the 20th century, 3 influenza viruses caused major pandemics: the 1918 H1N1 virus, the 1957 H2N2 virus, and the 1968 H3N2 virus. These pandemics were initiated by the introduction and successful adaptation of a novel hemagglutinin subtype to humans from an animal source, resulting in antigenic shift. Despite global concern regarding a new pandemic influenza, the emergence pathway of pandemic strains remains unknown. Here we estimated the evolutionary history and inferred date of introduction to humans of each of the genes for all 20th century pandemic influenza strains. Our results indicate that genetic components of the 1918 H1N1 pandemic virus circulated in mammalian hosts, i.e., swine and humans, as early as 1911 and was not likely to be a recently introduced avian virus. Phylogenetic relationships suggest that the A/Brevig Mission/1/1918 virus (BM/1918) was generated by reassortment between mammalian viruses and a previously circulating human strain, either in swine or, possibly, in humans. Furthermore, seasonal and classic swine H1N1 viruses were not derived directly from BM/1918, but their precursors co-circulated during the pandemic. Mean estimates of the time of most recent common ancestor also suggest that the H2N2 and H3N2 pandemic strains may have been generated through reassortment events in unknown mammalian hosts and involved multiple avian viruses preceding pandemic recognition. The possible generation of pandemic strains through a series of reassortment events in mammals over a period of years before pandemic recognition suggests that appropriate surveillance strategies for detection of precursor viruses may abort future pandemics.     

Avian influenza and human health

Natural infections with influenza A viruses have been reported in a variety of animal species including humans, pigs, horses, sea mammals, mustelids and birds. Occasionally devastating pandemics occur in humans. Although viruses of relatively few HA and NA subtype combinations have been isolated from mammalian species, all 15 HA subtypes and all 9 NA subtypes, in most combinations, have been isolated from birds.In the 20th century the sudden emergence of antigenically different strains transmissible in humans, termed antigenic shift, has occurred on four occasions, 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977 (H1N1), each time resulting in a pandemic. Genetic analysis of the isolates demonstrated that 'new' strains most certainly emerged after reassortment of genes of viruses of avian and human origin in a permissive host. The leading theory is that the pig represents the 'mixing vessel' where this genetic reassortment may occur.In 1996, an H7N7 influenza virus of avian origin was isolated from a woman with a self-limiting conjunctivitis. During 1997 in Hong Kong, an H5N1 avian influenza virus was recognised as the cause of death of 6 of 18 infected patients. Genetic analysis revealed these human isolates of H5N1 subtype to be indistinguishable from a highly pathogenic avian influenza virus that was endemic in the local poultry population. More recently, in March 1999, two independent isolations of influenza virus subtype H9N2 were made from girls aged one to four who recovered from flu-like illnesses in Hong Kong. Subsequently, five isolations of H9N2 virus from humans on mainland China in August 1998 were reported. H9N2 viruses were known to be widespread in poultry in China and other Asian countries.In all these cases there was no evidence of human to human spread except with the H5N1 infections where there was evidence of very limited spread. This is in keeping with the finding that all these viruses possessed all eight genes of avian origin. It may well be that infection of humans with avian influenza viruses occurs much more frequently than originally assumed, but due to their limited effect go unrecognised.For the human population as a whole the main danger of direct infection with avian influenza viruses appears to be if people infected with an 'avian' virus are infected simultaneously with a 'human' influenza virus. In such circumstances reassortment could occur with the potential emergence of a virus fully capable of spread in the human population, but with antigenic characteristics for which the human population was immunologically naive. Presumably this represents a very rare coincidence, but one which could result in a true influenza pandemic.     

Risk of influenza A (H5N1) infection among health care workers exposed to patients with influenza A (H5N1), Hong Kong

The first outbreak of avian influenza A (H5N1) occurred among humans in Hong Kong in 1997. To estimate the risk of person-to-person transmission, a retrospective cohort study was conducted to compare the prevalence of H5N1 antibody among health care workers (HCWs) exposed to H5N1 case-patients with the prevalence among nonexposed HCWs. Information on H5N1 case-patient and poultry exposures and blood samples for H5N1-specific antibody testing were collected. Eight (3.7%) of 217 exposed and 2 (0.7%) of 309 nonexposed HCWs were H5N1 seropositive (P=.01). The difference remained significant after controlling for poultry exposure (P=.01). This study presents the first epidemiologic evidence that H5N1 viruses were transmitted from patients to HCWs. Human-to-human transmission of avian influenza may increase the chances for the emergence of a novel influenza virus with pandemic potential.     

Single gene reassortants identify a critical role for PB1, HA, and NA in the high virulence of the 1918 pandemic influenza virus

The 1918 influenza pandemic was exceptionally severe, resulting in the death of up to 50 million people worldwide. Here, we show which virus genes contributed to the replication and virulence of the 1918 influenza virus. Recombinant viruses, in which genes of the 1918 virus were replaced with genes from a contemporary human H1N1 influenza virus, A/Texas/36/91 (Tx/91), were generated. The exchange of most 1918 influenza virus genes with seasonal influenza H1N1 virus genes did not alter the virulence of the 1918 virus; however, substitution of the hemagglutinin (HA), neuraminidase (NA), or polymerase subunit PB1 genes significantly affected the ability of this virus to cause severe disease in mice. The 1918 virus virulence observed in mice correlated with the ability of 1918 recombinant viruses to replicate efficiently in human airway cells. In a second series of experiments, eight 1918 1:7 recombinants were generated, in which each Tx/91 virus gene was individually replaced by a corresponding gene from 1918 virus. Replication capacity of the individual 1:7 reassortant viruses was assessed in mouse lungs and human airway cells. Increased virus titers were observed among 1:7 viruses containing individual 1918 HA, NA, and PB1 genes. In addition, the 1918 PB1:Tx/91 (1:7) virus showed a distinctly larger plaque size phenotype than the small plaque phenotype of the 1918 PA:Tx/91 and 1918 PB2:Tx/91 1:7 reassortants. These results highlight the importance of the 1918 HA, NA, and PB1 genes for optimal virus replication and virulence of this pandemic strain.