Infectious entry pathway of influenza virus in a canine kidney cell line

The entry of fowl plague virus, and avian influenza A virus, into Madin- Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 degrees C, viruses bound to the cell surface but were not internalized. Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37 degrees C, part of the virus became resistant to removal b neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium. The neuraminidase- resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0 degrees C and, after warming at 37 degrees C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles. Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures. At physiological pH, no fusion of the virus with the plasma membrane was observed. When prebound virus was incubated at a pH of 5.5 or below for 1 min at 37 degrees C, fusion was, however, detected by ferritin immunolabeling. t low multiplicity, 90% of the prebound virus became neuraminidase- resistant and was presumably fused after only 30 s at low pH. These experiments suggest that fowl plague virus enters MDCK cells by endocytosis in coated pits and coated vesicles and is transported to the lysosome where the low pH initiates a fusion reaction ultimately resulting in the transfer of the genome into the cytoplasm. The entry pathway of fowl plague virus thus resembles tht earlier described for Semliki Forest virus.     

Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics

The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377-8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.     

Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector.

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has played a major role in studies on the molecular biology of insect DNA viruses. Recently, this system has been effectively adapted as a highly efficient vector in insect cells for the expression of several mammalian genes. A cDNA sequence of the influenza (fowl plague) virus haemagglutinin gene has been inserted into the BamHI site of the pAc373 polyhedrin vector. Spodoptera frugiperda cells were co-transfected with this construct, pAc-HA651, and authentic AcNPV DNA. Recombinant virus was selected by adsorption of transfected cells to erythrocytes followed by serial plaque passages on S. frugiperda cells. We have determined the site of insertion of the haemagglutinin gene into the AcNPV genome by restriction enzyme cleavage and Southern blot hybridization analyses using haemagglutinin cDNA as a probe. The influenza haemagglutinin gene is located in the polyhedrin gene of AcNPV DNA. Immunofluorescent labelling, immunoprecipitation and immunoblot analyses with specific antisera revealed that S. frugiperda cells produce immune reactive haemagglutinin after infection with the recombinant virus. The haemagglutinin is expressed at the cell surface and has haemolytic capacity that has been activated by post-translational proteolytic cleavage. When chickens were immunized with S. frugiperda cells expressing haemagglutinin, they developed haemagglutinin-inhibiting and neutralizing antibodies and were protected from infection with fowl plague virus. These observations demonstrate that the haemagglutinin is processed in insect cells in a similar fashion as in fowl plaque virus-infected vertebrate cells and that it has full biological activity.     

Genetic recombination for antigenic markers of antigenically different strains of influenza B virus

Incorporation of trypsin in agar overlay or fluid maintenance media resulted in enhancement of plaquing efficiency and replication of influenza B viruses in primary chicken embryo fibroblasts. Using this improved technique, recombination was attempted with two serologically distinct strains of influenza B virus, B/Lee/40 and B/Massachusetts/1/71. After mixed infection, two virus clones were selected and characterized in detail. Hemagglutination inhibition and neuraminidase inhibition tests showed that these viruses are reciprocal antigenic recombinants with hemagglutinin derived from one parent and neuraminidase from the other. Serological examinations of the antisera to these recombinants confirmed the results. The frequency of recombination was high in the present system and 64% of the virus clones isolated without selection from the mixed yield were recombinants. This high recombination frequency is consistent with the genomic reassortment that is characteristic of recombination of influenza A viruses.     

Identification of host genes linked with the survivability of chickens infected with recombinant viruses possessing H5N1 surface antigens from a highly pathogenic avian influenza virus

Seventeen recombinant viruses were generated by a reverse genetic technique to elucidate the pathogenicity of highly pathogenic avian influenza viruses (HPAIVs) in chickens. The recombinant viruses generated possessed hemagglutinin (HA) and neuraminidase (NA) genes from an HPAIV. Other segments were combinations of the genes from an HPAIV and two low-pathogenic avian influenza viruses (LPAIVs) derived from chicken (LP) and wild bird (WB). Exchange of whole internal genes from an HPAIV with those of an LPAIV resulted in a significant extension of the survival time following intranasal infection of the chickens with the recombinants. Survival analysis demonstrated that the exchange of a gene segment affected survivability of the chickens with statistical significance. The analysis revealed three groups of recombinants with various gene constellations that depended upon the survivability of the infected chickens. Recombinants where the PA gene was exchanged from LP to WB in the LP gene background, LP (W/PA), did not kill any chickens. LP (W/PA) replicated less efficiently both in vitro and in vivo, suggesting that the intrinsic replication ability of LP (W/PA) affects pathogenicity; however, such a correlation was not seen for the other recombinants. Microarray analysis of the infected chicken lungs indicated that the expression of 7 genes, CD274, RNF19B, OASL, ZC3HAV1 [corrected] , PLA2G6, GCH1, and USP18, correlated with the survivability of the chickens infected (P < 0.01). Further analysis of the functions of these genes in chickens would aid in the understanding of host gene responses following fatal infections by HPAIVs.     

Evaluation of the ESPLINE INFLUENZA A&B-N Kit for the diagnosis of avian and swine influenza

The ESPLINE INFLUENZA A&B-N kit was evaluated for its applicability to the rapid diagnosis of influenza in chickens and pigs. The kit specifically detected viral antigens in tracheal swabs and tissue homogenates of the trachea, liver, spleen, and colon of chickens inoculated with a highly pathogenic avian influenza virus strain, A/chicken/Yamaguchi/7/04 (H5N1), at 48 hr post-inoculation (p.i.) as well as in the tracheal and cloacal swabs and tissue homogenates of dead chickens. For those infected with a low pathogenic strain, A/chicken/aq-Y-55/01 (H9N2), antigens were detected only in the samples from tracheal swabs and organs 1-4 days p.i. The kit also detected viral antigens in the nasal swabs of miniature pigs infected with swine and avian influenza viruses. The kit was found to be sensitive and specific enough for the rapid diagnosis of infections of influenza A virus in chickens and pigs.     

Highly pathogenic H5N1 influenza virus causes coagulopathy in chickens

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus-infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.     

Mapping of the influenza virus genome. III. Identification of genes coding for nucleoprotein, membrane protein, and nonstructural protein.

In previous communications we reported that the eight RNA segments of influenza A/PR/8/34 (HON1) virus could be distinguished from corresponding segments of influenza A/Hong Kong/8/68 (H3N2) virus by migration on polyacrylamide-urea gels. Examination of the RNA patterns of the two parent viruses and recombinants derived from them in concert with serological identification of surface proteins and analysis of the other proteins on sodium dodecyl sulfate gradient gels permitted the identification of the genes coding for hemagglutinin, neuraminidase, and the P1, P2, and P3 proteins (Palese and Schulman, 1976; P. Palese et al., Virology, in press). In the present report we have extended these observations using similar techniques to examine other recombinants and have identified the genes coding for the remaining virus-specific moving RNA segment as 1) and segment 6 of Hong Kong virus coding for the respective nucleoproteins, and that segment 7 of both viruses codes for the membtane protein and RNA segment 8 codes for the nonstructural protein. This completes the mapping of the influenza A virus genome.     

Nucleoprotein and membrane protein genes are associated with restriction of replication of influenza A/Mallard/NY/78 virus and its reassortants in squirrel monkey respiratory tract.

An avian influenza A virus, A/Mallard/NY/6750/78(H2N2), was restricted in in replication in the respiratory tract of squirrel monkeys. Avian-human influenza A reassortant viruses possessing the six RNA segments coding for nonsurface proteins (i.e., internal genes) of this avian virus were as restricted in replication in squirrel monkeys as their avian influenza parent. These findings indicated that restriction of replication of the avian influenza virus is a function of one or more of its internal genes. For an investigation of which of the avian influenza genes was responsible for restricted replication in the respiratory tract of primates, reassortant viruses were produced that contained human influenza virus surface antigens from the A/Udorn/72(H3N2) virus and one or more of the internal genes derived from the avian influenza virus parent. Avian-human reassortant influenza A viruses containing only the nucleoprotein or matrix protein RNA segment from the avian influenza virus parent were as restricted in their growth as an avian-human influenza reassortant virus containing each of the six avian influenza internal genes. In addition, an avian-human influenza reassortant virus possessing only the avian RNA 1 and nonstructural genes (which by themselves do not specify restricted replication) manifested a significant reduction of virus replication in squirrel monkey tracheas. Thus, the avian nucleoprotein and matrix genes appear to play a major role in the host range restriction exhibited by the A/Mallard/78 virus and its reassortants, but the combination of RNA 1 and nonstructural genes also contributes to restriction of replication.     

Acid stability of the hemagglutinin protein regulates H5N1 influenza virus pathogenicity

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.     

Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport.

The influenza A/fowl plague virus/Rostock/34 hemagglutinin (HA), which is cleaved intracellularly and has a high pH threshold (pH 5.9) for undergoing its conformational change to the low-pH form, was expressed from cDNA in CV-1 and HeLa T4 cells in the absence of other influenza virus proteins. It was found, by biochemical assays, that the majority of the HA molecules were in a form indistinguishable from the low-pH form of HA. The acidotropic agent, ammonium chloride, stabilized the accumulation of HA in its native form. Coexpression of HA and the homotypic influenza virus M2 protein, which has ion channel activity, stabilized the accumulation of HA in its pH neutral (native) form, and the M2 protein ion channel blocker, amantadine, prevented the rescue of HA in its native form. These data provide direct evidence that the influenza virus M2 protein ion channel activity can affect the status of the conformational form of cleaved HA during intracellular transport.     

Avian influenza virus

Recent outbreaks of highly pathogenic avian influenza in chickens and ducks that occurred in 9 Asian countries including Japan alarmed to realize that there is no border for infections and gave a rise to great concern for human health as well as for agriculture. This H5N1 virus jumped the species barrier and caused severe disease with high mortality in humans in Viet Nam and Thailand; 15 deaths of 22 cases and 8 of 12, respectively. A second concern was the possibility that the situation could give rise to another influenza pandemic in humans since genetic reassortment may occur between avian and human influenza viruses when a person is concurrently infected with viruses from both species. This process of gene swapping inside the human body can give rise to a new subtype of the influenza virus to which humans would not have immunity. The outbreaks also emphasized the need to continue active surveillance on avian influenza throughout the year to undertake aggressive emergency control measures as soon as an infection is detected.     

Chemotherapy and vaccination: a possible strategy for the control of highly virulent influenza virus.

The influenza A virus [A/Chicken/Pennsylvania/1370/83 (H5N2)] that caused up to 80% mortality among chickens provided a model system for testing the efficacy of chemotherapeutic agents against highly virulent influenza virus. Amantadine and rimantadine administered in drinking water were efficacious both prophylactically and therapeutically. However, under conditions simulating natural transmission of virus, amantadine- and rimantadine-resistant viruses arose and were transmitted to other birds in contact with the infected chickens, causing mortality. Simultaneous administration of inactivated H5N2 vaccine and amantadine provided protection. Thus, chemotherapy may be useful in the treatment of a highly pathogenic influenza virus outbreak in humans or other animals when used in combination with vaccine.     

Selection and identification of influenza virus recombinants of defined genetic composition.

The RNAs of influenza virus recombinants were analyzed on polyacrylamide gels under conditions in which the derivation of specific RNA segments (including those coding for hemagglutinin and neuraminidase) could be determined. Analysis of the RNAs of recombinant viruses with identical hemagglutinin and neuramindase revealed that the derivation of the remaining genes could be influenced by UV irradiation of one of the parent viruses. In five of seven such recombinants all of the remaining identifiable genes were derived from the nonirradiated parent, whereas in two others only the three largest RNA segments were derived from the nonirradiated parent. Analysis of the RNA pattern of a recombinant isolated from mixed infection in which neither parent was irradiated demonstrated a random mixture of RNA segments derived from the two parent viruses.     

Development of an immunochromatographic kit for rapid diagnosis of H5 avian influenza virus infection

Highly pathogenic avian influenza (HPAI) caused by the H5N1 subtype has given rise to serious damage in poultry industries in Asia. The virus has expanded its geographical range to Europe and Africa, posing a great risk to human health as well. For the control of avian influenza, a rapid diagnosis by detecting the causative virus and identifying its subtype is essential. In the present study, a rapid diagnosis kit combining immunochromatography with enzyme immunoassay which detects the H5 HA antigen of influenza A virus was developed using newly established anti-H5 HA monoclonal antibodies. The present kit specifically detected all of the H5 influenza viruses tested, and did not react with the other HA subtypes. H5 HA antigens were detected from swabs and tissue homogenates of chickens infected with HPAI virus strain A/chicken/Yamaguchi/7/04 (H5N1) from 2 days post inoculation. The kit showed enough sensitivity and specificity for the rapid diagnosis of HPAI.     

The NS1 gene contributes to the virulence of H5N1 avian influenza viruses

In the present study, we explored the genetic basis underlying the virulence and host range of two H5N1 influenza viruses in chickens. A/goose/Guangdong/1/96 (GS/GD/1/96) is a highly pathogenic virus for chickens, whereas A/goose/Guangdong/2/96 (GS/GD/2/96) is unable to replicate in chickens. These two H5N1 viruses differ in sequence by only five amino acids mapping to the PA, NP, M1, and NS1 genes. We used reverse genetics to create four single-gene recombinants that contained one of the sequence-differing genes from nonpathogenic GS/GD/2/96 and the remaining seven gene segments from highly pathogenic GS/GD/1/96. We determined that the NS1 gene of GS/GD/2/96 inhibited the replication of GS/GD/1/96 in chickens, while the substitution of the PA, NP, or M gene did not change the highly pathogenic properties of GS/GD/1/96. Conversely, of the recombinant viruses generated in the GS/GD/2/96 background, only the virus containing the NS1 gene of GS/GD/1/96 was able to replicate and cause disease and death in chickens. The single-amino-acid difference in the sequence of these two NS1 genes resides at position 149. We demonstrate that a recombinant virus expressing the GS/GD/1/96 NS1 protein with Ala149 is able to antagonize the induction of interferon protein levels in chicken embryo fibroblasts (CEFs), but a recombinant virus carrying a Val149 substitution is not capable of the same effect. These results indicate that the NS1 gene is critical for the pathogenicity of avian influenza virus in chickens and that the amino acid residue Ala149 correlates with the ability of these viruses to antagonize interferon induction in CEFs.     

Human and murine cytotoxic T cells specific to respiratory syncytial virus recognize the viral nucleoprotein (N), but not the major glycoprotein (G), expressed by vaccinia virus recombinants

The viral antigens recognized by cytotoxic T cells (CTL) have not been defined in most viruses infecting mouse or man. Natural or artificial virus recombinants can be used to determine the antigen specificity of CTL directed against viruses with segmented genomes, such as influenza, but this technique is more difficult to apply to the study of unsegmented viruses. We describe here the use of recombinant vaccinia viruses, containing cDNA corresponding to either the nucleoprotein (N) gene or the major surface glycoprotein (G) gene of human respiratory syncytial virus (RSV), to examine the antigen specificity of anti-RSV cytotoxic T cells from humans and mice. The results demonstrate that the RSV N protein is one of the target antigens for CTL in man and mouse, whereas the G protein was not recognized and can at best represent a minor target antigen for CTL.     

Generation of seal influenza virus variants pathogenic for chickens, because of hemagglutinin cleavage site changes.

Influenza virus A/seal/Mass/1/80 (H7N7) was adapted to grow in MDCK cells and chicken embryo cells (CEC) in the absence of exogenous protease. The biological properties of the virus variants obtained coincided with intracellular activation of the hemagglutinin (HA) by posttranslational proteolytic cleavage and depended on the cell type used for adaptation. MDCK cell-adapted variants contained point mutations in regions of the HA more distant from the cleavage site. It is proposed that these mutations are probably responsible, through an unknown mechanism, for enhanced cleavability of HA in MDCK cells. Such virus variants were apathogenic in chickens. CEC-adapted variants, on the other hand, contained an insertion of basic amino acids at the HA cleavage site, in addition to scattered point mutations. The insertions converted the cleavage sites in the variant virus HAs so that they came to resemble the cleavage site found in highly pathogenic avian influenza viruses. CEC variants with such cleavage site modifications were highly pathogenic for chickens. The lethal outcome of the infection in chickens demonstrated for the first time that an influenza virus derived from a mammalian species can be modified during adaptation to a new cell type to such an extent that the resulting virus variant becomes pathogenic for an avian species.     

Membrane fusion activity of influenza virus.

A simple assay is described to monitor fusion between fowl plague virus (FPV, an avian influenza A virus) and liposomes which allows the simultaneous quantitation of both lytic and non-lytic fusion events. As in fusion between viruses and the plasma membrane and in FPV-induced cell-cell fusion, the reaction only occurs at pH 5.5 or below, and it is fast, highly efficient, and essentially non-lytic when fresh virus and liposomes are used. The fusion occurs over a broad temperature range, and has no requirement for divalent cations. The fusion factor of influenza virus is a hemagglutinin (HA) spike which protrudes from the virus membrane and which is also responsible for virus binding to the host cell. The finding that fusion occurs as efficiently with liposomes containing or lacking virus receptor structures, further emphasizes the remarkable division of labor in the HA molecule: the receptor-binding sites are located in the globular HA1 domains and the fusion activation peptide is found at the N-terminal of HA2 in the stem region of the protein. The mechanism of fusion is discussed in terms of the three-dimensional structure of the HA and the conformational change which the protein undergoes at the fusion pH optimum.