Crystallization and peptide maps of neuraminidase “heads” from H2N2 and H3N2 influenza virus strains

Neuraminidase “heads” of two H2N2 influenza virus strains (A/Nederlands/68 and A/Korea/68) and one H3N2 strain (A/Hong Kong/68) were isolated by pronase digestion of recombinant viruses possessing neuraminidase molecules from these strains and H (NWS) hemagglutinin. The neuraminidase heads were purified and crystallized by dialysis against distilled water. Large flat crystals, which differed in appearance between the three strains, were obtained. Tryptic peptide maps of the crystallized neuraminidase heads of the Nederlands/68 and Hong Kong/68 strains were almost identical, suggesting that a virus closely related to the Nederlands/68 (H2N2) strain may have been the source of the neuraminidase for the formation of the H3N2 Hong Kong subtype.     

Antineuraminidase antibody response to vaccination of chickens with intact virus and different subunit preparations of the influenza virus strains A/Sing/1/57 (H2N2), A/Hong Kong/1/68 (H3N2) and A/Port Chalmers/1/73 (H3N2)

Summary The antineuraminidase (AN) antibody response to vaccination of chickens with intact virus and different subunit preparations of the influenza virus strains A/Sing/1/57 (H2N2), A/Hong Kong/1/68 (H3N2) and A/Pt. Chalmers/1/73 (H3N2) was tested comparatively.Using a photometric method capable of analysing mixtures of AN antibodies against antigenically different N2 neuraminidases, it was concluded that vaccination with subunits produced by treatment with bromelain and Sarkosyl can yield AN antibody response against heterologous neuraminidase. By contrast, vaccination with intact and ether-treated virus gave AN antibody response against homologous neuraminidase, only.These findings were confirmed when testing sera by means of enzyme inhibition test and by adsorption experiments with homologous and heterologous neuraminidases.The conclusion was reached that the NA's of the strains A/Sing/1/57 and A/Pt. Chalmers/1/73 share antigenic determinants and that the NA of the strain A/Hong Kong/1/68 shares antigenic determinants with that of the strains A/Sing/1/57 and A/Pt. Chalmers/1/73.Abbreviations used ACU antibody concentration unit - AN antineuraminidase - HA hemagglutinin - HCU hemagglutinin concentration unit - mU micro units - NA neuraminidase - NIT neuraminidase inhibition test - PBS 0.15 M NaCl solution buffered at pH 7.0 with 0.01 M phosphate A portion of the experiments described in this paper has been presented at the International Symposium on Animal and Human Influenzas, Alfort, September 1979.     

Failure of attenuated temperature-sensitive influenza A (H3N2) virus to induce heterologous interference in humans to parainfluenza type 1 virus.

The present investigation was undertaken to determine if a candidate live vaccine virus, influenza A/Hong Kong/68-ts-1 [E] (H3N2), induced heterologous interference against an interferon-sensitive, wild-type, parainfluenza type 1 challenge virus. The parainfluenza virus was administered 7 days after Hong Kong/68-ts-1 [E] virus infection. The clinical response, daily quantitative virus shedding, interferon production, and serum and nasal wash antibody responses were determined in an experimental group (influenza A virus followed by parainfluenza virus) and 10 volunteers in a control group (parainfluenza virus only). The volunteers were selected on the basis of susceptibility to the two viruses, i.e. serum hemagglutination-inhibition antibody titer of is less than or greater to 1:8 for influenza virus and low nasal wash antibody titer (is less than or greater to 1:8) for parainfluenza virus. Despite a 100% infection rate in the Hong Kong/68-ts-1 [E] vaccinees, no heterologous interference was induced against the parainfluenza type 1 virus challenge.     

Influenza virus A/Anas acute/Maritime Territory/730/76(H3N2) isolated from wild ducks in the Maritime Territory

A strain A/Anas acuta/Primorie/730/76 was isolated in the autumn of 1976 from wild pintails in the Primorskiy kray, USSR, and found by the H1 and neuraminidase activity inhibition test to be antigenically related to the reference A/Hong Kong/1/68 strain. The CFT showed the A/Anas acuta/Primorie/730/76 strain to contain in its hemagglutinin 3 antigenic determinants: two common with the A/Hong Kong/1/68 strain and one the strain-specific, lacking in the human influenza viruses of the H3N2 antigenic formula.     

Studies on antigenic variation in influenza virus. Evidence for multiple antigenic determinants on the hemagglutinin subunits of A-Hong Kong-68 (H3 N2) virus and the A-England-72 strains

Hemagglutinin subunits were isolated from Hong Kong influenza virus (A/Hong Kong/68, H3 N2) and from its antigenic variants, A/England/42/72, and A/Memphis/102/72 which arose as the result of antigenic drift. Immunodiffusion tests showed that the HK/68 subunits possessed at least two different kinds of antigenic determinants while the 1972 variants appeared to possess at least three different determinants. Evidence suggested that the different antigenic determinants were located on the same hemagglutinin subunit and that the virus particles did not possess a mixture of antigenically different subunits.One of the antigenic determinants was common to the hemagglutinin subunits of A/Hong Kong/68, A/England/42/72 and A/Memphis/102/72 viruses. Antibody to this determinant cross-reacted in immunodiffusion, hemagglutination-inhibition, and neutralization tests with all three viruses. Antibodies to another determinant showed no detectable serological cross-reactions between HK/68 and Mem/72 viruses. Thus it appeared that during antigenic drift, Hong Kong influenza virus had undergone an antigenic shift in this “specific” determinant.Antibody to the “specific” determinant on HK/68 hemagglutinin (which did not react at all with Mem/72) did, however, show some cross-reaction with A/England/42/72 hemagglutinin suggesting that this virus was an intermediate in the evolution of the Memphis/72 variant.Despite the occurrence of an antigenic shift in one determinant, peptide maps of the light polypeptides (HA2) from the hemagglutinin subunits of HK/68 and Mem/72 viruses were almost identical and maps of the heavy polypeptides (HA1) showed only a relatively small number of differences.     

Studies on the origin of pandemic influenza: III. Evidence implicating duck and equine influenza viruses as possible progenitors of the Hong Kong strain of human influenza

Two strains of influenza virus isolated from horses and ducks in 1963, A/equine/Miami/1/63 (Heq2 Neq2) and A/duck/Ukraine/1/63 (Hav7 Neq2) were found to possess hemagglutinin subunits which cross-reacted in hemagglutination-inhibition and immunodiffusion tests with those of the Hong Kong strain of human influenza A/Hong Kong/1/68 (H3 N2).Peptide maps of the heavy polypeptide chains from the hemagglutinin subunits of these three strains showed a number of differences, but maps of the light chains were almost identical, indicating that the light polypeptide chains from the hemagglutinin subunits of these animal, avian and human viruses had practically the same amino acid sequence.One explanation of these results is that the three viruses arose, by genetic recombination, from a common ancestor.     

The inoculative properties of cold-adapted reassortant A(H5N2) influenza strain during intranasal administration to mice

Classical genetic reassortant techniques were used to have a cold-adapted (ca) reassortant A/17/Duck/Potsdam/86/92 (H5N2) that inherited the hemagglutinin (HA) gene from the nonpathogenic avian virus A/Duck/Potsdam/ 1402-6186 (H5N2) and the genes of neuraminidase (NA) and non-glycated proteins from the ca attenuation donor A/Leningrad/134/17/57 (H2N2). All experiments were performed under increased biological protection (BSV-3+). The reassortant and parent H5N2 virus were non-pathogenic to Balb/c mice, the reassortant replication in the murine nasal passages (3.5 Ig EID50/ml) being higher than that in the lung (2.1 lg EID50/ml). Intranasal inoculation of mice with reassortant A/17/Duck/Potsdam/86/92 caused an immune response to both homological H5N2 virus and antigenically differing variants of influenza A (H5N1) virus isolated from humans in 1997 and 2003. The mice intranasally immunized with the ca reassortant were protected against fatal infection with the highly pathogenic A/Hong Kong/483/9797 (H5N1) virus and against infection with A/Hong Kong/213/03(H5N1) virus (80 and 100%, respectively).     

Comparative analysis of the dynamics of antibodies to influenza A (H1N1) and A/Hong Kong/1/68 (H3N2) in the sera of a healthy population

A comparative analysis of the dynamics of antibodies against A(H1N1) and A/Hong Kong/I/68 (H3N2) influenza viruses was carried out in the blood sera of healthy population in some cities of the USSR. The data obtained suggest that anamnestic antibody production can be stimulated only by the drift- and not by the shift-variants of viruses, i.e. by those with varying hemagglutinin serotypes.     

Characterization of a 1980-swine recombinant influenza virus possessing H1 hemagglutinin and N2 neuraminidase similar to that of the earliest Hong Kong (H3N2) virus

A recombinant (H1N2, formerly Hsw 1N2), A/swine/Ehime/1/80 was found to possess antigenic, biological and genomic characteristics different from those of a previous A/swine/Kanagawa/2/78 (H1N2) strain. Five monoclonal antibodies to A/NJ/8/76 definitely differentiated the hemagglutinin molecules of the former virus from the latter, showing that these viruses differed, at least, at two antigenic determinants. Neuraminidase-inhibition tests with monoclonal antibodies to different H2N2 and H3N2 viruses revealed that the A/swine/Ehime/1/80 strain contained a neuraminidase very similar to that of the late human Asian (H2N2) and the earliest Hong Kong (H3N2) viruses. Growth comparison of swine and human isolates indicated that A/swine/Ehime/1/80 and A/swine/Shizuoka/1/78 (H1N1) failed to grow at 42 degrees C, while A/swine/Kanagawa/2/78 and its possible parental virus, A/swine/Kanagawa/4/78 (H1N1) replicated efficiently at this stringent temperature. These results revealed that the viruses having growth characteristics similar to those of avian influenza virus were present in the swine population. RNA analysis by oligonucleotide mapping suggested that A/swine/Ehime/1/80 may be a recombinant between A/swine/Shizuoka/1/78-like and A/Aichi/2/68 (H3N2)-like viruses. To further determine the gene constellation of this recombinant virus, DNA-RNA hybridization was performed by using DNA segments complementary for swine (H1N1) virus RNA and the entire RNAs of three viruses. The molecular hybridization could define the genomic composition of the recombinant, indicating that only the neuraminidase gene of this virus is derived from the earliest Hong Kong (H3N2)-like virus and remaining seven genes from swine (H1N1) virus.     

A pandemic H1N1 influenza virus-like particle vaccine induces cross-protection in mice

Influenza virus-like particles (VLPs) represent promising alternative vaccines. However, it is necessary to demonstrate that influenza VLPs confer cross-protection against antigenically distinct viruses. In this study, a VLP vaccine comprising hemagglutinin (HA) and M1 from the A/California/04/2009 (H1N1) were used and its ability to induce cross-protective efficacy against heterologous viruses A/PR/8/34 (H1N1) and A/New Caledonia/20/99 (H1N1) in mice was assessed. Vaccination with 2009 H1 VLPs induced significantly higher levels of IgG cross-reactive with these heterologous viruses after the second boost compared to after the prime or first boost. Lung virus titers also decreased significantly and the lung cross-reactive IgG response after lethal virus challenge was significantly greater in immunized mice compared to naïve mice. Vaccinated mice showed 100% protection against A/PR/8/34 and A/Caledonia/20/99 viruses with only moderate body weight loss and induction of cross-reactive recall, IgG antibody-secreting cell responses. The variations in HA amino acid sequences and antigenic sites were determined and correlated with induction of cross-protective immunity. These results indicate that VLPs can be used as an effective vaccine that confers cross-protection against antigenically distinct viruses.     

Preparative isolation of A/Hong Kong/1/68 (H3N2) virus hemagglutinin]

Hemagglutinin of A/Hong Kong/1/68 virus was isolated by electrophoresis in acetate-cellulose of a recombinant 12/13 (H3N1) strain destroyed by sodium dodecyl sulphate. Electron microscope examinations were carried out and molecular weights of hemagglutinin polypeptides were determined. A monospecific serum containing no antibody to neuraminidase was prepared.     

Serological evidence of natural recombinant influenza virus (Hsw1N2) infection among pigs in Japan

A new recombinant influenza A virus (Hsw1N2) infection among hogs in Miyagi prefecture, Japan, was confirmed by serological examinations. Nineteen out of twenty sera collected in one farm in January 1979 showed high haemagglutination inhibition antibody titres to Hsw1 antigen (A/New Jersey/8/76) and also significant neuraminidase inhibition antibody titres to N2 antigen of the Hong Kong 1973 variant (A/Port Chalmers/1/73) but not to N1 antigen. The results indicate the occurrence of genetic reassortment between Hsw1N1 and H3N2 viruses in Japanese pigs and spread of the recombinant in nature.     

Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong.

Analysis of the sequences of all eight RNA segments of the influenza A/G oose/Guangdong/1/96 (H5N1) virus, isolated from a sick goose during an outbreak in Guangdong province, China, in 1996, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5N1 viruses isolated in Hong Kong in 1997. However, the remaining genes showed greater similarity to other avian influenza viruses. Notably, the neuraminidase gene did no have the 19-amino-acid deletion in the stalk region seen in the H5N1 Hong Kong viruses and the NS gene belonged to allele B, while that of the H5N1 Hong Kong viruses belonged to allele A. These data suggest that the H5N1 viruses isolated from the Hong Kong outbreaks derived their HA genes from a virus similar to the A/Goose/Guangdong/1/96 virus or shared a progenitor with this goose pathogen.     

Potency of an inactivated influenza vaccine prepared from A/duck/Hong Kong/960/1980 (H6N2) against a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice

H6 influenza viruses are prevalent in domestic and wild birds in Eurasian countries and have been isolated from pigs and a human. To prepare for an influenza pandemic, we have established an influenza virus library consisting of more than 1,300 influenza virus strains, including 144 combinations of 16 hemagglutinin and 9 neuraminidase subtypes. H6 viruses in the library were classified into Early, Group II, Group III, and W312 sublineages and the North America lineage on the basis of their phylogenetic features. Chicken antisera to A/duck/Hong Kong/960/1980 (H6N2) of the Early sublineage broadly reacted with viruses of different sublineages in a hemagglutinin inhibition test. A whole inactivated virus particle vaccine was prepared from A/duck/Hong Kong/960/1980 (H6N2) which was stocked in the influenza virus library. The potency of this vaccine against A/duck/Vietnam/OIE-0033/2012 (H6N2), which belongs to a different sublineage, was evaluated in mice. The test vaccine was sufficiently potent to induce an immune response that reduced the impact of disease caused by a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice. The present results indicate that the whole inactivated virus particle vaccine prepared from a virus strain in the influenza virus library is useful as a vaccine against pandemic influenza.     

Phylogenetic Analysis of Hemagglutinin and Neuraminidase Genes of H9N2 Viruses Isolated from Migratory Ducks

Genetic analysis indicated that the pandemic influenza strains derived from wild aquatic birds harbor viruses of 15 hemagglutinin (HA) and 9 neuraminidase (NA) antigenic subtypes. Surveillance studies have shown that H9N2 subtype viruses are worldwide in domestic poultry and could infect mammalian species, including humans. Here, we genetically analyzed the HA and NA genes of five H9N2 viruses isolated from the migratory ducks in Hokkaido, Japan, the flyway of migration from Siberia during 1997–2000. The results showed that HA and NA genes of these viruses belong to the same lineages, respectively. Compared with those of A/quail/Hong Kong/G1/97-like and A/duck/Hong Kong/Y280/97-like viruses, HA and NA of the migratory duck isolates had a close relationship with those of H9N2 viruses isolated from the chicken in Korea, indicating that the Korea H9N2 viruses might be derived from the migratory ducks. The NA genes of the five isolates were located in the same cluster as those of N2 viruses, which had caused a human pandemic in 1968, indicating that the NA genes of the previous pandemic strains are still circulating in waterfowl reservoirs. The present results further emphasize the importance of carrying out molecular epidemiological surveillance of H9N2 viruses in wild ducks to obtain more information for the future human influenza pandemics preparedness.     

Radioimmunological analysis of the neuraminidase of influenza virus subtype N2

Competitive radioimmunoassay in the homologous system using 125I-labeled neuraminidase (NA) of A/Japan/305/57 virus and antibodies to it showed that influenza viruses NA of subtypes H2N2 (A/Singapore/1/57 and A/Tokyo/3/67) and H3N2 (A/Aichi/2/68, A/England/42/72, A/Port Chalmers/1/73, and A/Texas/1/77) had undergone clear antigenic change despite the presence of Japan strain NA determinant. The use of a heterologous system (125I-NA of the Japan strain--antibodies to NA of England/42/72 strain) for the study of the intratype determinant showed the presence of a common determinant in all the strains under study, this determinant being represented dissimilarly, however. Neuraminidase of the Texas strain has a determinant differing from the intratype one, common for the Japan and England viruses.     

Polygenic virulence factors involved in pathogenesis of 1997 Hong Kong H5N1 influenza viruses in mice

Virulence factors of influenza A (H5N1) viruses collected in 1997 from mammalian hosts were examined using a BALB/c mouse model. Fifteen amino acid (aa) residues in four influenza virus genes which correlated with high- and low-pathogenic phenotypes in mice were identified by analyzing sequence alignments. In addition to these specific residues, the effects of aa residue 627 of the PB2 gene, and the hemagglutinin (HA) and neuraminidase (NA) genes were also investigated using a reverse genetics system established with representative viruses of low (A/Hong Kong/486/97) and high (A/Hong Kong/483/97) pathogenicity for mice. None of 15 aa residues alone had any effect on virulence. The HA and NA genes had a synergistic effect on virulence and the absence of a glycosylation site at aa154 in the HA gene also increased virulence of virus. Multiple genes are involved in virulence of Hong Kong H5N1 influenza A viruses for mice with the presence of lysine at aa627 in the PB2 gene exhibiting a significantly larger effect than the HA and NA genes.     

Antigenic determinants of influenza virus hemagglutinin--IX. The carbohydrate side chains from an Asian strain

The heavy chain (HA1) of hemagglutinin from the Asian variant A/Japan/305/57 was examined to establish which of the four oligosaccharide units on this polypeptide could cross-react with host antigen. Cyanogen bromide and enzymic digests were fractionated and the resulting peptides screened for their ability to bind to antibodies raised against uninfected chick chorioallantoic membranes. This 324 residue HA1 polypeptide from the Asian strain A/Japan/305/57 is known to contain oligosaccharide units at asparagine residues 11, 23, 285 and at either 164, 165 or both. The peptides carrying the four carbohydrate units have been isolated, the monosaccharide compositions determined and the location of the fourth oligosaccharide shown to be Asn164 by direct amino acid sequence analysis. All four sugar units on A/Japan/305/57 HA1 are of the N-acetyllactosamine type, but only the one attached to Asn11 could cross-react with anti-(host antigen) antibodies. This antigenic carbohydrate unit on the Asian hemagglutinin heavy chain is located in a similar region to the two antigenically active oligosaccharides located on the hemagglutinin from the Hong Kong variant A/Memphis/102/72. The study was therefore extended to include the early Hong Kong variant A/Aichi/2/68, which has the same oligosaccharide distribution as A/Memphis/102/72 but different sugar compositions for the individual oligosaccharide units Like A/Memphis/102/72, only the N-acetyllactosamine units attached at positions 8 and 22 of A/Aichi/2/68 were capable of binding to anti-(host antigen) IgG. Finally, the light chain (HA2) polypeptides from A/Bel/42 (HO), the A/Japan/305/57 (H2) and the two Hong Kong variants A/Aichi/2/68 (X-31) and A/Memphis/102/72 were also examined and found to contain carbohydrate which is antigenically related to chick embryo host antigen.     

Genome-sequenee Analysis of the Pathogenic H5N1 Avian Influenza A Virus Isolated in China in 2004

Analysis of the sequences of the genome of the avian influenza A/chicken/Hubei/327/2004 (H5N1) virus, isolated from a poultry farm during the outbreak of avian influenza (AI) in Hubei Province, central China, in the spring of 2004, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5 highly pathogenic avian influenza virus (HPAI). Notably, the neuraminidase gene of the virus had a 20-amino acid deletion in the stalk region and a 5-amino acid deletion in the NS gene which belonged to allele B. Furthermore, the internal genes (PB2, PA, NP, M2) of the A/chicken/Hubei/327/2004 virus with the particular amino acid residues were more closely related to H5N1 viruses of 2000–2003 isolated in Hong Kong and the AIV of Thailand and Vietnam in 2004, but less likely to evolve from the viruses of Hong Kong 1997. Finally, our results demonstrated that the influenza A/chicken/Hubei/327/2004 (H5N1) virus was similar to those of the AI viruses isolated from Hong Kong (2000–2003), Vietnam, and Thailand rather than the viruses from the 1997 lineage of Hong Kong and with closest genetic relatives to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus. These data suggest that the influenza A/chicken/Hubei/327/2004 (H5N1) virus which circulated in central China derived its internal gene from a virus similar to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus.     

Annual examination of influenza virus infection among pigs in Miyagi prefecture, Japan: the appearance of Hsw1N1 virus.

Annual dissemination of swine and Hong Kong influenza viruses among hogs in Miyagi prefecture, Japan, was examined by virus recovery and serological tests. The serological examination revealed that two distinct influenza A viruses, H3N2 and Hsw1N1, had caused dual epidemic in pigs since mid-1977. Some individual sera contained antibodies against both strains. Although positive sera against Hong Kong or swine influenza viruses appeared in the serosurvey throughout the year, the positive ratios indicated a peak in the early spring of 1978. During this high prevalence period, positive sera against the H3N2 strain were found throughout the prefecture, but high incidences of Hsw1N1 virus were found only in limited areas to the north-east of Sendai City. Four strains of influenza A virus were isolated from bronchial swabs collected from hogs in April, 1978. These strains were identified as Hsw1N1 viruses and were closely related to the A/New Jersey/8/76 strain.