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The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed ("functional"), or unlicensed ("hypofunctional"). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m-/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab')2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.

The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.

Influenza A virus (IAV) non-structural protein 1 (NS1) has multiple functions, is essential for virus replication and may be a good target for IAV diagnosis. To generate broadly cross-reactive NS1-specific mAbs, mice were immunized with A/Hong Kong/1/1968 (H3N2) 6×His-tagged NS1 and hybridomas were screened with glutathione S-transferase-conjugated NS1 of A/Puerto Rico/8/1934 (H1N1). mAbs were isotyped and numerous IgG-type clones were characterized further. Most clones specifically recognized NS1 from various H1N1 and H3N2 IAV types by both immunoblot and immunofluorescence microscopy in mouse M1, canine Madin-Darby canine kidney and human A549 cells. mAb epitopes were mapped by overlapping peptides and selective reactivity to the newly described viral NS3 protein. These mAbs detected NS1 in both the cytoplasm and nucleus by immunostaining, and some detected NS1 as early as 5 h post-infection, suggesting their potential diagnostic use for tracking productive IAV replication and characterizing NS1 structure and function. It was also demonstrated that the newly identified NS3 protein is localized in the cytoplasm to high levels.

The first confirmed outbreak of highly pathogenic avian influenza (HPAI) virus infections in North America was caused by A/turkey/Ontario/7732/1966 (H5N9); however, the phylogeny of this virus is largely unknown. This study performed genomic sequence analysis of 11 avian influenza isolates from 1956 to 1979 for comparison with A/turkey/Ontario/7732/1966 (H5N9). Phylogenetic and genetic analyses included these viruses in combination with all known full-genome sequences of avian viruses isolated before 1981. It was shown that a low-pathogenic avian influenza virus, A/turkey/Ontario/6213/1966 (H5N1), that had been isolated 3 months previously, was the closest known genetic relative with six genome segments of common lineage encoding the polymerase subunits PB2, PB1 and PA, nucleoprotein (NP), haemagglutinin (HA) and non-structural (NS) proteins. The lineages of these genome segments included reassortment with other North American turkey viruses that were all rooted in North American wild waterfowl with the HA gene originating from the H5N2 serotype. The phylogenies demonstrated adaptation from North American wild birds to turkeys with the possible involvement of domestic waterfowl. The turkey isolate, A/turkey/Wisconsin/1968 (H5N9), was the second most closely related poultry isolate to A/turkey/Ontario/7732/1966 (H5N9), possessing five common lineage genome segments (PB2, PB1, PA, HA and neuraminidase). The A/turkey/Ontario/6213/1966 (H5N1) virus was more virulent than A/turkey/Wisconsin/68 (H5N9) for chicken embryos and mice, indicating a greater biological similarity to A/turkey/Ontario/7732/1966 (H5N9). Thus, A/turkey/Ontario/6213/1966 (H5N1) was identified as the closest known ancestral relative of HPAI A/turkey/Ontario/7732/1966 (H5N9), which will serve as a useful reference virus for characterizing the early genetic and biological properties associated with the emergence of pathogenic avian influenza strains.

The role of the NS1 protein in modulating influenza A virulence and host range was assessed by adapting A/Hong Kong/1/1968 (H3N2) (HK-wt) to increased virulence in the mouse. Sequencing the NS genome segment of mouse-adapted variants revealed 11 mutations in the NS1 gene and 4 in the overlapping NEP gene. Using the HK-wt virus and reverse genetics to incorporate mutant NS gene segments, we demonstrated that all NS1 mutations were adaptive and enhanced virus replication (up to 100 fold) in mouse cells and/or lungs. All but one NS1 mutant was associated with increased virulence measured by survival and weight loss in the mouse. Ten of twelve NS1 mutants significantly enhanced IFN-β antagonism to reduce the level of IFN β production relative to HK-wt in infected mouse lungs at 1 day post infection, where 9 mutants induced viral yields in the lung that were equivalent to or significantly greater than HK-wt (up to 16 fold increase). Eight of 12 NS1 mutants had reduced or lost the ability to bind the 30 kDa cleavage and polyadenylation specificity factor (CPSF30) thus demonstrating a lack of correlation with reduced IFN β production. Mutant NS1 genes resulted in increased viral mRNA transcription (10 of 12 mutants), and protein production (6 of 12 mutants) in mouse cells. Increased transcription activity was demonstrated in the influenza mini-genome assay for 7 of 11 NS1 mutants. Although we have shown gain-of-function properties for all mutant NS genes, the contribution of the NEP mutations to phenotypic changes remains to be assessed. This study demonstrates that NS1 is a multifunctional virulence factor subject to adaptive evolution.

Adaptive evolution is characterized by positive and parallel, or repeated selection of mutations. Mouse adaptation of influenza A virus (IAV) produces virulent mutants that demonstrate positive and parallel evolution of mutations in the hemagglutinin (HA) receptor and non-structural protein 1 (NS1) interferon antagonist genes. We now present a genomic analysis of all 11 genes of 39 mouse adapted IAV variants from 10 replicate adaptation experiments. Mutations were mapped on the primary and structural maps of each protein and specific mutations were validated with respect to virulence, replication, and RNA polymerase activity. Mouse adapted (MA) variants obtained after 12 or 20-21 serial infections acquired on average 5.8 and 7.9 nonsynonymous mutations per genome of 11 genes, respectively. Among a total of 115 nonsynonymous mutations, 51 demonstrated properties of natural selection including 27 parallel mutations. The greatest degree of parallel evolution occurred in the HA receptor and ribonucleocapsid components, polymerase subunits (PB1, PB2, PA) and NP. Mutations occurred in host nuclear trafficking factor binding sites as well as sites of virus-virus protein subunit interaction for NP, NS1, HA and NA proteins. Adaptive regions included cap binding and endonuclease domains in the PB2 and PA polymerase subunits. Four mutations in NS1 resulted in loss of binding to the host cleavage and polyadenylation specificity factor (CPSF30) suggesting that a reduction in inhibition of host gene expression was being selected. The most prevalent mutations in PB2 and NP were shown to increase virulence but differed in their ability to enhance replication and demonstrated epistatic effects. Several positively selected RNA polymerase mutations demonstrated increased virulence associated with >300% enhanced polymerase activity. Adaptive mutations that control host range and virulence were identified by their repeated selection to comprise a defined model for studying IAV evolution to increased virulence in the mouse.

Background: Influenza viruses cause serious infections that can be prevented or treated using vaccines or antiviral agents, respectively. While vaccines are effective, they have a number of limitations, and influenza strains resistant to currently available anti-influenza drugs are increasingly isolated. This necessitates the exploration of novel anti-influenza therapies., Methodology/principal Findings: We investigated the potential of aurintricarboxylic acid (ATA), a potent inhibitor of nucleic acid processing enzymes, to protect Madin-Darby canine kidney cells from influenza infection. We found, by neutral red assay, that ATA was protective, and by RT-PCR and ELISA, respectively, confirmed that ATA reduced viral replication and release. Furthermore, while pre-treating cells with ATA failed to inhibit viral replication, pre-incubation of virus with ATA effectively reduced viral titers, suggesting that ATA may elicit its inhibitory effects by directly interacting with the virus. Electron microscopy revealed that ATA induced viral aggregation at the cell surface, prompting us to determine if ATA could inhibit neuraminidase. ATA was found to compromise the activities of virus-derived and recombinant neuraminidase. Moreover, an oseltamivir-resistant H1N1 strain with H274Y was also found to be sensitive to ATA. Finally, we observed additive protective value when infected cells were simultaneously treated with ATA and amantadine hydrochloride, an anti-influenza drug that inhibits M2-ion channels of influenza A virus., Conclusions/significance: Collectively, these data suggest that ATA is a potent anti-influenza agent by directly inhibiting the neuraminidase and could be a more effective antiviral compound when used in combination with amantadine hydrochloride.

H3-subtype influenza viruses are known to infect avian and mammalian species, including humans. However, little is known about the prevalence of H3 influenza virus infection in chicken populations in China. Therefore, a serologic survey of chickens was conducted in China to investigate the seroprevalence of avian H3-subtype influenza virus. Anti-H3 antibodies were assayed by using hemagglutination inhibition (HI) and confirmatory virus neutralization (VN) testing of 4598 serum samples, collected between July 2006 and June 2007, from 173 chicken flocks located in 18 areas that included 16 provinces and two municipalities. Seroepidemiologic results indicated that avian H3-subtype viruses were circulating in chickens in some regions of China, regions that included 12 of the 18 test areas, with an overall average prevalence rate of 2.83%. Samples from 44 of 173 flocks were HI/VN seropositive, including 15 flocks with levels that ranged from 10.00% to 41.94%. Significantly higher seroprevalence rates were observed in older chicken flocks and in those sampled in the cooler seasons. Standardized comparisons showed that Guangdong and Jiangsu, located in the south and east of China, respectively, had significantly higher levels of H3 seropositivity. For the first time, these results demonstrated serologic evidence for H3 avian influenza virus infection in chicken populations in several locations throughout China. These observations highlight the need for continued epidemiologic surveillance of the H3 subtype and for other low-pathogenic avian influenza viruses in China and other regions.

Influenza A virus (FLUAV) reverse genetics requires the cloning of all eight viral genome segments into genomic expression plasmids using restriction enzyme cleavage and ligation. Herein is described the construction of a pair of plasmid vectors and their use in RecA Escherichia coli for direct recombination with influenza cDNA for reverse genetics. This approach is simpler; avoiding restriction digestion and ligation while maintaining the required orientation of genome segments. For this recombinational approach two plasmid constructs were generated, pHH21A and pHH21G, that both possess a 25 nucleotide recombination cassette comprised of the consensus 5' and 3' ends of the negative strand divided by a StuI cleavage site, but that differ at position 4 from the 3' end due to the presence of an A or G nucleotide (plus sense) to correspond to differences among genome segments. Using the described procedure it was possible to clone viral cDNA genomes of several avian and human FLUAVs into genomic expression plasmids in a single recombination step. This novel approach to generating sets of genomic plasmid constructs for reverse genetics reduces the time and complexity of procedures thus avoiding complications that would delay rescue of viral genomes for vaccine production or biological characterization and analysis.

A rapid and effective lateral flow assay (LFA) for detection of avian influenza virus (AIV) was developed. For antigen capture, the assay used monoclonal antibody specific for a conserved nuclear protein (NP) epitope, immobilized on a cellulose acetate matrix, in conjunction with a second NP monoclonal antibody chemically linked to either coloured latex beads or colloidal gold particles contained in a sample pad for detection. Virus sample added to the sample pad flowed into the trapping antibody to form a visible band as well as a second, control band further along the acetate strip. The control band consisted of recombinant protein A/G, also immobilized on the matrix. A second LFA for detection of chicken antibody to AIV was developed where NP antigen was immobilized on the matrix with recombinant protein A/G immobilized as a control band. Latex beads or colloidal gold particles to which monoclonal anti-chicken antibody was attached, were used as the indicator system.

Rationale: Studies have shown that cigarette smoke impacts respiratory host defense mechanisms; however, it is poorly understood how these smoke-induced changes impact the overall ability of the host to deal with pathogenic agents., Objective: The objective of this study was to investigate the impact of mainstream cigarette smoke exposure on immune inflammatory responses and viral burden after respiratory infection with influenza A., Methods: C57BL/6 mice were sham- or smoke-exposed for 3 to 5 mo and infected with either 2.5 x 10(3) pfu (low dose) or 2.5 x 10(5) pfu (high dose) influenza virus., Measurements and Main Results: Although smoke exposure attenuated the airway's inflammatory response to low-dose infection, we observed increased inflammation in smoke-exposed compared with sham-exposed mice after infection with high-dose influenza, despite a similar rate of viral clearance. The heightened inflammatory response was associated with increased expression of tumor necrosis factor-alpha, interleukin-6, and type 1 IFN in the airway, and increased mortality. Importantly, smoke exposure did not interfere with the development of influenza-specific memory responses; sham- and smoke-exposed animals were equally protected upon viral rechallenge., Conclusion: Our study suggests that, in mice, cigarette smoke affects primary antiviral immune-inflammatory responses, whereas secondary immune protection remains intact.

Recent human infections caused by the highly pathogenic avian influenza virus H5N1 strains emphasize an urgent need for assessment of factors that allow viral transmission, replication, and intra-airway spread. Important determinants for virus infection are epithelial cell receptors identified as glycans terminated by an alpha2,3-linked sialic acid (SA) that preferentially bind avian strains and glycans terminated by an alpha2,6-linked SA that bind human strains. The mouse is often used as a model for study of influenza viruses, including recent avian strains; however, the selectivity for infection of specific respiratory cell populations is not well described, and any relationship between receptors in the mouse and human lungs is incompletely understood. Here, using in vitro human and mouse airway epithelial cell models and in vivo mouse infection, we found that the alpha2,3-linked SA receptor was expressed in ciliated airway and type II alveolar epithelial cells and was targeted for cell-specific infection in both species. The alpha2,6-linked SA receptor was not expressed in the mouse, a factor that may contribute to the inability of some human strains to efficiently infect the mouse lung. In human airway epithelial cells, alpha2,6-linked SA was expressed and functional in both ciliated and goblet cells, providing expanded cellular tropism. Differences in receptor and cell-specific expression in these species suggest that differentiated human airway epithelial cell cultures may be superior for evaluation of some human strains, while the mouse can provide a model for studying avian strains that preferentially bind only the alpha2,3-linked SA receptor.

Uncoating of influenza occurs in endosomes where the acid environment is instrumental in membrane fusion and the dissociation of the ribonucleoprotein (RNP) from matrix protein by the action of the hemagglutinin and M2 protein ion channels, respectively. Earlier studies have shown that low pH treatment results in the release of M1 protein from RNP. To obtain RNP free of M1 protein, we attempted to isolate RNP by velocity sedimentation on pH 5 glycerol gradients; however, the RNP sedimented as pellets under centrifugation conditions that had previously resolved RNP on neutral gradients. The increase in sedimentation rate occurred between pH 5.6 and 6.0 and was reversible for a portion of the RNP on raising the pH to neutrality. RNP isolated from infected cells or virions sedimented on acidification and was seen to form clumps visible by electron microscopy. If acidification preceded NP40 detergent lysis, virion RNP appeared to be released as genomic complexes. The pH threshold for viral membrane fusion was 5.8 indicating that the same pH condition also resulted in aggregation of RNP. Because exposure of virions to pH 5 occurs during uncoating in endosomes and is essential for infectivity, it is possible that low pH-induced RNP aggregation may facilitate aspects of viral uncoating such as dissociation of RNP from M1 or transport of genomes to the nucleus.

The genetic basis for virulence in influenza virus is largely unknown. To explore the mutational basis for increased virulence in the lung, the H3N2 prototype clinical isolate, A/HK/1/68, was adapted to the mouse. Genomic sequencing provided the first demonstration, to our knowledge, that a group of 11 mutations can convert an avirulent virus to a virulent variant that can kill at a minimal dose. Thirteen of the 14 amino acid substitutions (93%) detected among clonal isolates were likely instrumental in adaptation because of their positive selection, location in functional regions, and/or independent occurrence in other virulent influenza viruses. Mutations in virulent variants repeatedly involved nuclear localization signals and sites of protein and RNA interaction, implicating them as novel modulators of virulence. Mouse-adapted variants with the same hemagglutinin mutations possessed different pH optima of fusion, indicating that fusion activity of hemagglutinin can be modulated by other viral genes. Experimental adaptation resulted in the selection of three mutations that were in common with the virulent human H5N1 isolate A/HK/156/97 and that may be instrumental in its extreme virulence. Analysis of viral adaptation by serial passage appears to provide the identification of biologically relevant mutations.

Adaptation of the prototype A/FM/1/47 H1N1 strain to mice resulted in selection of the A/FM/1/47-MA variant with increased virulence. Earlier analysis identified mutations in the HA and M1 genes that increase virulence in the mouse. Complete sequence analysis identified mutations in the PB1, PB2, HA, NA, and M1 genes. Reassortants were produced between the parental FM and FM-MA strains to obtain viruses that differ due to combinations of mutant genes. To assess the relationship between virulence and replication, the median lethal dose was determined for mice and growth properties were assessed in mouse lung, MDCK cells and chicken embryo. Not only were all five mutations shown to control virulence but also the replicative capacity in the mouse. The HA, NA and M1 mutations increased yield in all three hosts whereas in combination the PB1 and PB2 mutations were host restrictive changing the virus to a mouse specific strain. For the NA and M1 mutations the increase in growth in mouse lung was proportional to a 2-fold (log10) increase in virulence however the HA mutation increased virulence largely independent of increased growth indicating a change in pathological properties that damage the host. Thus mutations that affect virulence can be classified according to host-dependent and independent ability to increase growth as well as changes in pathological properties. Each of the PB1, PB2, NA, HA, and M1 genes acquired gain-of-function mutations for mouse infection that involve structural motifs that may serve as markers for virulence or targets for antiviral therapy.

Mouse-adapted influenza A virus, FM-MA, interferes with the replication of wild-type strains on co-infection. The interference phenotype was previously mapped to FM-MA segment 2 encoding a mutant PB1 protein, the catalytic component of the RNA polymerase complex. To identify the point at which FM-MA interferes with wild-type A/HK/1/68 (HK), the relative levels of transcription and genome replication of the PB1, NP and M1 genes were determined for FM-MA and HK viruses in co-infected cells using RT-PCR. All stages of HK macromolecular synthesis (primary and secondary transcription, genomic RNA, complementary RNA and protein synthesis) were suppressed relative to FM-MA. Infection with HK virus alone resulted in the accumulation of similar or greater amounts of RNA at late times post-infection relative to FM-MA thus indicating that the presence of FM-MA specifically compromised HK transcription and replication in co-infected cells. However early in infection FM-MA was ten times more active in mRNA transcription than HK or its parental strain FM. FM-MA's ability to interfere was primarily due to an increased capacity for primary transcription. FM-MA genomes were also selectively assembled into progeny virus from cells co-infected with HK and FM-MA, a step which was distinct from the capacity for enhanced RNA synthesis. This suggests that interference of HK growth by FM-MA in mixed infections results from two distinct events: a preferential synthesis of FM-MA-specific macromolecules which is then augmented by a preferential assembly of FM-MA genomes.

The mouse adapted strain of influenza A/FM/1/47 virus, FM-MA, has increased virulence due to mutations in HA, M1 and at least one other, unmapped, genome segment. Genetic reassortants that differ due to the HA or M1 mutations were used to define the role of these mutations in pathogenesis. Pathological changes in lungs of infected mice were assessed by hematoxylin phloxine saffron (HPS) staining, and viral infection was measured by fluorescent antibody staining of thin sections and flow cytometry of lung parenchymal cells. HA played a role in bronchiolar pathology by increasing necrosis of bronchiolar epithelium, peribronchiolar lymphocytes, and airway obstruction. The HA mutation was shown to be responsible for a 0.2 unit decreased in the pH optimum of fusion and controlled resistance to alpha and beta inhibitors of hemagglutination. Both these changes in biology may confer a replicative advantage in bronchioles seen in the first day of infection. Thus the HA mutation may have conferred a survival advantage in the extracellular lung environment. The M1 mutation resulted in improved growth in the lung and cultured cells and was associated with increases in recruitment of macrophages, spread of infection into the alveoli of the lung and interstitial pneumonia. Sequence analysis indicated that the unmapped mutation in the control of FM-MA virulence is either the K482-->R substitution in the PB2 protein or the D538-->G substitution in the PB1 protein. One or other of these mutations results in a growth advantage in infected lung but not in cultured cells as well as a further increased recruitment and infection of macrophages in the lung. Infection with virulent strains of influenza that induced increases in macrophage recruitment caused hypothermia in the mouse.

Genetic analysis of mouse-adapted influenza virus variant A/FM/1/47 (FM) MA has previously identified four genome segments, 4, 5, 7, and 8, that are statistically associated with virulence. On sequencing these genome segments, we found single amino acid replacements at amino acid 47 of the HA2 subunit of the hemagglutinin and at amino acid 139 of the matrix protein. Mutation was not detected in segments 5 and 8, obviating a role for these genes in FM-MA virulence. FM-MA replicates to higher titer than FM in MDCK cells and in mouse lung. FM X FM-MA reassortants were used to show that the M1 gene controlled replication in MDCK cells as well as in mouse lung.

On mouse adaption of A/FM/1/47, a variant, A/FM/1/47-MA (FM-MA), that had acquired the properties of increased virulence and interference was produced. Coinfection of cells with FM-MA and prototype strains of influenza virus yielded > 100-fold more FM-MA virus than prototype virus, whereas coinfection with the same prototype strains and the parental A/FM/1/47 virus produced equivalent yields, indicating that FM-MA had acquired mutations that confer the property of interference during mouse adaption. FM-MA is a nondefective interfering virus that grows to a high titer in vivo and in vitro. It has previously been shown that segments 4, 7, and 8 and possibly segment 5 account for the increased virulence. In this study we show by genetic analysis of FM-MA x A/HK/1/68 reassortants that segment 2, coding for the polymerase-associated protein PB1, and possibly segment 8, encoding the NS1 and NS2 proteins, control the ability of FM-MA to interfere. Interference could not be overcome by increasing the titer of the coinfecting strain, but delaying FM-MA infection by 4 to 6 h did avoid interference. During interference of A/HK/1/68, protein synthesis was inhibited by less than 65% throughout coinfection. Given the kinetics of interference and the small perturbation in protein synthesis, interference appeared to occur at the level of late genome replication or virus assembly. Virulence and interference in FM-MA were not linked. An interfering avirulent FM-MA x A/HK/1/68 reassortant, E07, was capable of protecting mice against lethal pneumonia due to a virulent noninterfering reassortant, H04.

To cause disease, influenza virus must possess several genetically determined abilities that mediate stages in pathogenesis. The virulent mouse-adapted variant A/FM/1/47-MA (FM-MA), derived from the avirulent A/FM/1/47 (FM) strain, had acquired mutations in genes that control virulence. The purpose of this study was to identify those genes that had mutated to result in increased virulence and to obtain viruses that differed in virulence because of differences in individual genome segments. The genes that had mutated to increase virulence were initially identified by genetic analysis of reassortants obtained by crossing FM-MA with the avirulent strain A/HK/1/68 (HK). FM-MA genome segments 4, 5, 7, and 8 were significantly associated with virulence, as determined by using the Wilcoxon ranked sum analysis. The role of FM-MA segments 4, 7, and 8 was confirmed by reintroduction of these genes into the parental strain, which also provided virus strains that differed in virulence because of mutations in individual genome segments. Segments 4, 7, and 8 were responsible for a 10(3.6)-fold increase in virulence that was proportioned 10(2.2)-, 10(0.7)-, and 10(0.8)-fold, respectively. The role of segment 5 could not be confirmed on transfer back into the parental strain because of reversion during preparation of such reassortants. The incidence of reversion was shown to be significantly associated with culturing of FM-MA in chicken embryo cells but was not associated with growth in MDCK cells. The genetic analysis of FM-MA suggests that adaptation to increased virulence is an incremental process that involves the acquisition of mutations in multiple genes, each of which plays an individual role in pathogenesis. The structural and functional properties of segments 4, 7, and 8 that control the virulence of FM-MA can now be determined by using viruses that differ in virulence because of mutations in these individual genome segments.

Influenza undergoes extensive antigenic variation in nature. These new antigenic variants invariably supplant the preceding antigenic type that then disappears. In apparent contrast to this, two H3N2 strains that were forwarded to the Laboratory Centre for Disease Control, Ottawa, in 1984 from a Canadian public health laboratory for reference analysis were shown to be most closely related to 1973 and 1974 strains. Laboratory contamination on isolation by the regional public health laboratory was the most likely explanation for the occurrence of these strains, since one of the isolates (RV/76/84) was identical by T1 mapping to an A/Eng-like isolate being grown in the laboratory of isolation. The two isolates, RV/74/84 and RV/76/84, were shown to be distinct from each other and from prototype H3N2 strains by RNase T1 oligonucleotide mapping, SDS-PAGE, peptide mapping of hemagglutinin, and hemagglutination inhibition assay. RV/74 and RV/76 appeared to have been genetically stable for the 10 to 11 years preceding 1984; this is consistent with laboratory frozen storage for this period of time. This paper demonstrates the utility of RNase T1 mapping for the characterization of novel or spurious influenza isolates.

The first isolate of influenza virus in Canada during the winter of 1986 to 1987 was a genetic variant of A/Taiwan/1/86. This genetic variant type was the predominant strain obtained from several of the western provinces. The variant strains were antigenically indistinguishable from A/Taiwan/1/86 but were remarkably distinct by T1 oligonucleotide mapping. T1 mapping of individual genome segments indicated that the variants evolved from an A/Taiwan/1/86-like virus through the accumulation of point mutation or deletion or insertion events and probably do not contain foreign genes. The relative distribution of genetic variation was approximately equal among the individual genes, with the possible exception of segments 1 or 2 that were analyzed in combination and thus could not be individually associated with the observed variation.