Isolation of a genotypically unique H5N1 influenza virus from duck meat imported into Japan from China

An H5N1 influenza A virus was isolated from duck meat processed for human consumption, imported to Japan from Shandong Province, China in 2003. This virus was antigenically different from other H5 viruses, including the Hong Kong H5N1 viruses isolated from humans in 1997 and 2003. Sequence analysis revealed that six genes (PB1, PA, HA, NA, M, and NS) of this virus showed >97% nucleotide identity with their counterparts from recent H5N1 viruses, but that the remaining two genes (PB2 and NP) were derived from other unknown viruses. This duck meat isolate was highly pathogenic to chickens upon intravenous or intranasal inoculation, replicated well in the lungs of mice and spread to the brain, but was not as pathogenic in mice as H5N1 human isolates (with a dose lethal to 50% of mice (MLD50)=5x10(6) 50% egg infectious doses [EID50]). However, viruses isolated from the brain of mice previously infected with the virus were substantially more pathogenic (MLD50=approximately 10(2) EID50) and possessed some amino acid substitutions relative to the original virus. These results show that poultry products contaminated with influenza viruses of high pathogenic potential to mammals are a threat to public health even in countries where the virus is not enzootic and represent a possible source of influenza outbreaks in poultry.     

Isolation of an influenza A virus from seals

Summary Influenza A virus of serotype Hav1 Neq1 (H7N7 by the 1980 revised influenza typing system proposed by WHO experts) was repeatedly isolated from lung and brain tissues taken from harbor seals (Phoca vitulina) found suffering from pneumonia on Cape Cod Peninsula (U.S.A.) in the winter of 1979–1980. The seal isolates, although of a serotype identical to some fowl plague virus strains, were harmless to chickens and turkeys in transmission experiments. An earlier human infection by a Hav1 Neq1 influenza virus and the serologic relatedness of this avian serotype with the equine 1 serotype are cited in support of the view that influenza viruses with these antigenic characteristics seem to have a facility to pass from birds to mammals.     

Isolation of a type A influenza virus from an Australian pelagic bird

A type A influenza virus was isolated from a tracheal swab taken from an apparently healthy shearwater bird (mutton bird, Puffinus pacificus chlororhynchus) nesting on Tryon Island off the east coast of Australia. The hemagglutinin subunits of the shearwater virus were of antigenic subtype Hav6, but the neuraminidase subunits were not related antigenically to those of any known virus and represent a new neuraminidase subtype, Nav5. The hemagglutinin and neuraminidase subunits of the shearwater virus were segregated by recombination of the virus with other influenza A viruses. The size and polypeptide composition of these subunits, isolated electrophoretically from the SDS-disrupted recombinant viruses, were similar to those of other influenza viruses.These findings suggest that there may be other avian influenza viruses with novel hemagglutinin or neuraminidase subunits, from which human pandemic strains could arise by genetic recombination.     

Isolation of an influenza H1N1 virus from a pig

A type A influenza virus was isolated from a pig in 1979 in Thailand. Hemagglutinin and neuraminidase antigens of this virus were similar to those of human H1N1 virus isolated also in Thailand shortly before. Genetic relatedness between the human and swine viruses was demonstrated by the electrophoretic pattern of RNA on urea-polyacrylamide gel and by oligonucleotide mapping of RNA, suggesting that the swine virus had been derived from human H1N1 virus.     

Isolation of a recombinant influenza virus (Hsw1N2) from swine in Japan

Summary Outbreaks of swine influenza were first observed in Japan in 1978. A number of influenza viruses were isolated from diseased swine. Almost all viruses isolated were swine influenza virus (Hsw1N1) but two viruses isolated from the nasal swabs of swine showing clinical signs of influenza in the Kanagawa prefecture were characterized antigenically as Hsw1N2. Analysis of swine sera showed that influenza virus Hsw1N2 was epidemic in the farm from which the virus had been isolated. The new virus (Hsw1N2) seems to have been produced by recombination between swine influenza virus (Hsw1N1) and Hong Kong influenza virus (H3N2).     

Isolation of a highly pathogenic influenza virus from turkeys

An influenza virus was isolated from turkeys with an acute disease causing 30% mortality. The virus was subtyped as H5 N8. The nomenclature A/turkey/Ireland/83 (H5 N8) is proposed for this isolate. The virus had an ICPI of 1.80 to 1.85 for 1-day-old chicks and an IVPI of 2.74 for 6-week-old chickens. Following oronasal inoculation of juvenile and adult turkeys, chickens and ducks with the isolate, 100% mortality occurred in turkeys and chickens. No clinical signs were observed in inoculated ducks, but all developed serum antibody titres against the virus.     

Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion

The spike glycoprotein of influenza C/Johannesburg/1/66 was isolated in a soluble form by digestion of MDCK cell-grown virions with bromelain. The whole ectodomain of the glycoprotein could be recovered with an apparent molecular weight of 75,000 daltons determined in SDS-PAGE. Comparison to Triton X-100-isolated glycoprotein revealed that a C-terminal peptide of 3000-4500 daltons must have remained in the viral membrane. When purified by sucrose density gradient centrifugation the glycoprotein sedimented with a sedimentation coefficient of 10 S, indicating a molecular weight of 206,000 daltons, which is consistent with a trimeric structure of the spike molecule. The trimeric form was stabilized in sucrose gradients by Ca2+ ions. Bromelain digestion of virions with uncleaved glycoprotein, grown in MDCK cells without trypsin, produced two disulphide-linked subunits with similar electrophoretic mobilities in SDS-PAGE to the biologically active glycoprotein. The smaller subunit differed from the product cleaved in vivo (gp 30) by the presence of an additional arginine residue at the N-terminus. The soluble glycoprotein appears to possess both receptor-binding and receptor-destroying enzyme activities, as isolated glycoprotein inhibited hemagglutination of intact influenza C virions and showed RDE activity in an in vitro test. Glycoprotein exposed to low pH, which was sensitive to trypsin digestion, also demonstrated both these biological activities. Glycoprotein-mediated hemolysis could not be observed.     

Genetic analysis of an influenza B virus isolated from a patient with encephalopathy in Japan

An influenza B virus, B/Saga/S172/99 (SAG99), was isolated from the nasopharynx of a patient with encephalopathy/encephalitis in Japan in 1999. To clarify the molecular characteristics of this virus, detailed analysis of the gene segments coding for the hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M) and non-structural protein (NS) was undertaken. All five genes of SAG99 showed high nucleotide and predicted amino acid similarities with those of recent non-encephalopathic strains isolated in the same epidemic season. Subsequent phylogenetic analysis revealed that all five gene segments of SAG99 analyzed in the present study were most similar to those of the recent Yamagata/16/88-like viruses. The hemagglutinin and neuraminidase proteins of SAG99 were each distinguished from those of recent epidemic strains by one characteristic amino acid substitution. These substitutions were not found in the previously reported encephalopathy/encephalitis-derived influenza B viruses, and we could not find any common characteristic amino acid changes in SAG99 and these viruses. Similarly, among the internal proteins studied, only the M2 protein of SAG99 was found to contain a single novel amino acid change when compared with other recent isolates. Thus, it was apparent that SAG99 contained very few amino acid differences when compared with other epidemic viruses. The association of recent B/Yamagata/16/88-like viruses with encephalitis/encephalopathy observed in the present study and previously suggest that these viruses may have a higher potential for causing neurological complications in certain individuals.     

Isolation of an influenza A virus from ostriches (Struthio camelus)

An influenza A virus of the H7 N1 subtype was isolated from young ostriches which died after developing a syndrome characterized by a green discolouration of the urine, weakness and signs of respiratory distress. Mortality varied, depending on the age of the ostriches, the presence of other infectious agents and the amount of stress to which they were exposed. Using the haemagglutination inhibition test, an amnestic response was recorded in ostriches which recovered from the disease. Pathogenicity tests indicated that the isolate was of low virulence for chickens.     

Structural proteins of Getah virus isolates from Japan and Malaysia

Purified preparations of Getah virus strains have been analysed by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to reveal their structural proteins. Two envelope proteins (E1 and E2) and core protein (C) were found with the prototype AMM2021 strain both under reducing and nonreducing conditions, while separation of E1 and E2 was observed only under nonreducing conditions for 3 strains isolated in Japan. Limited digestion by Staphylococcus aureus V8 protease revealed difference in the peptide patterns of E1 between AMM2021 and Japanese isolates. Mobility of E1 and E2 was slower for the virus grown in BHK21 cells compared with the virus grown in Aedes albopictus cells, indicating host-controlled modification on the envelope glycoproteins.     

Absence of neuraminidase from influenza C virus

Summary Influenza C viruses did not possess neuraminidase activity when examined using either fetuin or sialyllactose as substrate. Purified preparations of influenza C virus inhibited hemagglutination by NWS hemagglutinin. The hemagglutination inhibiting activity was a bolished by treatment of influenza C virus with neuraminidase. These findings indicated the absence of neuraminidase activity on influenza C virus particles.With 2 Figures     

Isolation of preparative amounts of influenza virus hemagglutinin by an affinity chromatographic method

The use of affinity column sepharose-tyrosine-sulfanilic acid permits one to obtain preparative amounts of pure hemagglutinin of the types H1, H2, H3 from the total amount of surface glycoproteins solubilized by octylglycoside from antigenically different strains of influenza A virus. The yield of hemagglutinin ranges from 30% to 84% depending on the virus strain.     

Isolation and characterization of influenza C virus inhibitor in rat serum

Two hemagglutination inhibitors for influenza C virus were isolated from pooled sera of normal rats by sequential chromatography on Blue Sepharose CL 6B, Ultrogel AcA 22, and DEAE-cellulose. The two inhibitors were identified as alpha 1-macroglobulin and murinoglobulin by comparison with the authentic samples. These inhibitors abolished the hemagglutination by influenza C virus strains but did not affect the hemagglutination by influenza A and B virus strains. Hemagglutination inhibition activity of both inhibitors was completely destroyed by incubation with influenza C virus at 37 degrees C but not with the other types of influenza virus, indicating that the inhibitors are specific for influenza C virus. The inhibitory activity was also destroyed by incubation with neuraminidase from Arthrobacter ureafaciens. By contrast, no activity was lost after treatment with neuraminidase from Vibrio cholerae. These results suggest that the sialic acid residue(s) which is cleavable by the former neuraminidase but not by the latter is essential for the hemagglutination inhibition. The two inhibitors were inactivated by treating with sodium hydroxide and methylamine but not with sodium metaperiodate.     

Inhibition of influenza A virus replication by influenza B virus nucleoprotein: an insight into interference between influenza A and B viruses

Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP_FluB) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP_FluB suggest that functional NP_FluB is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP_FluB. Further analysis of NP_FluB indicated that it does not affect nuclear import of NP_FluA. Taken together, these findings suggest a novel role of NP_FluB in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.     

Isolation of influenza virus hemagglutinin and its separation into subunits by a stage-by-stage scheme for viral protein fractionation

The virus envelope protein, hemagglutinin, recovered by DTAB extraction of influenza virions, was purified to a high degree by the gel filtration high-performance liquid chromatography (GF HPLC). Then hemagglutinin was reduced and alkylated with iodoacetamide and the subunits were resolved by GF HPLC on the columns Spherogel TSK G 2000 SW and 3000 SW (600 X 7.5 mm ID).     

Genetic variation among human strains of influenza C virus isolated in Japan

The RNA genomes of sixteen human strains of influenza C virus isolated in Japan between 1964 and 1983 were compared by SDS-polyacrylamide gel electrophoresis and oligonucleotide fingerprinting. A high degree of genetic variation was observed among the strains analysed. However, there were some strains with the genomes closely related to one another, and they could be divided into two groups. The first group consists of C/Shizuoka/79, C/Kanagawa/1/81 and four strains of C/Yamagata/81. The 1981 strains of this group were all isolated in March of the year. The second one consists of C/Kyoto/41/82, C/Nara/82 and C/Hyogo/1/83 that were isolated between February 1982 and December 1983. Little or no difference was observed in the genomes of the same group, while the difference was evident between two groups. The Aichi/1/81 strain isolated in November 1981 had a genome distantly related to either of these two groups. Thus three different types of influenza C virus were isolated during the period of 12 mth from March 1981 to February 1982, suggesting that multiple influenza C viruses with distant genetic relationship were circulating at the same time in Japan.