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We constructed recombinant vaccinia viruses (RVV) expressing a 15-residue peptide (P18IIIB; RIQRGPGRAFVTIGK) of gp160 envelope protein from a human immunodeficiency virus type-1 (HIV-1) IIIB isolate using an H1 influenza virus hemagglutinin (HA) gene cassette. Immunofluorescent tests with antisera against both H1N1 influenza virus and P18IIIB localized chimeric HA molecules comprising influenza virus HA and P18IIIB peptide intracellularly, but the P18IIIB could not be seen on the outer surfaces of infected cells though weak fluorescence was detected regarding HA molecule. Consistent with these findings, Western blotting confirmed the expression of a polypeptide of about 74-kDa protein representing chimeric HA molecule in the infected cells. These recombinants markedly primed CD8+ cytotoxic T lymphocytes (CTL) specific for P18IIIB as well as HA protein of the influenza virus, but failed to elicit P18IIIB-specific antibody despite stimulating production of HA-specific antibody. In addition, the P18IIIB-specific CTL could strongly lyse target cells expressing the whole HIV-1 envelope gene of IIIB strain. Thus, the influenza virus chimeric HA cassette vector system used in the present study appeared to be a useful tool for constructing vaccine candidates which will predominantly prime CD8+ CTL specific for immunodominant determinants of various infectious agents. [ABSTRACT FROM AUTHOR]

Twenty-two strains of influenza A virus were isolated from caged birds which had been imported into Japan from India and Thailand and had died during transportation to Tokyo. Serological tests divided these strains into two groups. Viruses in the first group contained Hav7 hemagglutinin and were related antigenically to A/duck/ Ukraine/1/63 [Hav7 Neq2]; viruses in the second group contained Hav4 hemagglutinin and were related to A/duck/Czech/56 (Hav4 Nav1]. All strains contained Neq2 neuraminidase that was closely related to that of A/equine/Miami/1/63 [Heq2 Neq2] and A/duck/Ukraine/1/63 [Hav7 Neq2]. It was concluded that the strains in the first group were Hav7 Neq2 and those in the second group were Hav4 Neq2; both groups of viruses showed antigenic drift from the prototype strains. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We examined the characterization of the antiviral T lymphocytes elicited by immunization with a novel liposome vaccine (MDP-virosome) constructed with synthetic muramyldipeptide;[6-0-(2-tetradecylhexadecanoyl)-N-acetylmuramyl-L-alanyl-D-isoglutamine], cholesterol, influenza virus haemagglutinin and neuraminidase. The haemagglutinin glycoprotein first appeared to induce a significant subtype-specific cytotoxic activity through its arrangement on the inner and outer surfaces of the MDP-virosome. Splenocytes of BALB/c mice immunized with the virosome vaccine containing H3 haemagglutinin and N2 neuraminidase from human Hong Kong virus markedly lysed H3N2 virus-infected target cells, but not those infected with virus possessing a different subtype such as H1N1 surface antigens. Exposure of these splenic lymphocytes to virus antigen in vitro further enhanced their cytotoxic activity. The cytotoxic lymphocytes generated by the MDP-virosome vaccine expressed Thy 1 and CD4 antigens on their cell surface, and these activities were restricted by class II histocompatibility gene products. The marked reduction of pulmonary virus titres in infected mice caused by transferred immune spleen cells suggested that the MDP-virosome vaccination is able to protect against influenza virus infection through enhanced cellular immune responses. [ABSTRACT FROM AUTHOR]

New antigenic variants of B/Yamagata/16/88-like lineage which appeared in the season of 1997 as a minor strain tended to predominate in the following season. Also, we could observe for the first time, three peaks of activity caused by H3N2 virus and two variants of B influenza virus. Antigenic and phylogenetic analyses revealed that B/Victoria/2/87-like variants appeared again in Japan in 1997 after a nine-year absence. Influenza B viruses evolved into three major lineages, including the earliest strain (I), B/Yamagata/16/88-like variants (II), which comprised of three sublineages (II-(i), II-(ii), II-(iii)), and B/Victoria/2/87-like variants (III). Evolution of influenza B virus hemagglutinin was apparently distinguishable from that of influenza A virus, showing a systematic mechanism of nucleotide deletion and insertion. This phenomenon was observed to be closely related to evolutionary pathways of I, II-(i), II-(ii), II-(iii) and III lineages. It was noteworthy to reveal that the nucleotide deletion and insertion mechanism of influenza B virus completed one cycle over a fifty-year period, and that a three nucleotide deletion was again observed in 1997 strains belonging to lineage II-( iii). It was evident that amino acid substitutions accompanying nucleotide insertions were highly conserved. [ABSTRACT FROM AUTHOR]

Matrix (M) and nonstructural (NS) genes of thirteen equine H3N8 and H7N7 influenza viruses were sequenced and analyzed from an evolutionary point of view. The M and NS genes of H3N8 viruses isolated between 1989 and 1993 evolved into two minor branch clusters, including isolates from Europe and the American continent, respectively. It was noteworthy to reveal that the nucleotide sequences of the M and NS genes of an earlier American strain showed highest homology to those of recent European viruses. “Frozen evolution” was observed in the M and NS genes of A/eq/LaPlata/1/88. It was also evident that the NS gene of an H7N7 virus from 1977 was very similar to that of a 1979-H3N 8 virus, while the M gene was closest phylogenetically to that of the earliest H7N7 virus isolated in 1956. Furthermore, the M2 protein of A/eq/Newmarket/1/77 virus contained a carboxyl terminal deletion of three amino acids. The evolutionary rates of the M and NS genes of H3N8 equine influenza viruses were estimated to be 5.4 × 10−4 and 5.1 × 10−4 substitutions per site per year, respectively, which were slower than those of human viruses. [ABSTRACT FROM AUTHOR]

Recent human H3N2 influenza viruses isolated in Japan and China were characterised from an evolutionary point of view. They appeared to have divided into three minor branch clusters, including 1992-1993, 1993-1994 and 1994-1995 isolates. It was of particular interest to reveal that in addition to amino acid substitutions in the antigenic sites of the HA molecule, amino acid changes occurred at position 226 of the receptor binding site from lysine or glutamine to isoleucine in all strains belonging to the 1994-1995 branch cluster. This is the first evidence of human H3N2 influenza isolates, or any other influenza HA serotypes, to contain a conserved amino acid residue other than lysine or glutamine at this key position. [ABSTRACT FROM AUTHOR]

The nucleotide sequences of the HA1 domain of the H1 hemagglutinin genes of A/duck/Hong Kong/36/76, A/duck/Hong Kong/196/77, A/sw/North Ireland/38, A/sw/Cambridge/39 and A/Yamagata/120/86 viruses were determined, and their evolutionary relationships were compared with those of previously sequenced hemagglutinin (H1) genes from avian, swine and human influenza viruses. A pairwise comparison of the nucleotide sequences revealed that the genes can be segregated into three groups, the avian, swine and human virus groups. With the exception of two swine strains isolated in the 1930s, a high degree of nucleotide sequence homology exists within the group. Two phylogenetic trees constructed from the substitutions at the synonymous site and the third codon position showed that the H1 hemagglutinin genes can be divided into three host-specific lineages. Examination of 21 hemagglutinin genes from the human and swine viruses revealed that two distinct lineages are present in the swine population. The swine strains, sw/North Ireland/38 and sw/Cambridge/39, are clearly on the human lineage, suggesting that they originate from a human A/WSN/33-like variant. However, the classic swine strain, sw/Iowa/15/30, and the contemporary human viruses are not direct descendants of the 1918 human pandemic strain, but did diverge from a common ancestral virus around 1905. Furthermore, previous to this the above mammalian viruses diverged from the lineage containing the avian viruses at about 1880. [ABSTRACT FROM AUTHOR]

The nucleotide and deduced amino acid sequences of the haemagglutinin genes coding for the HA 1 domain of H3N8 equine influenza viruses isolated over wide regions of the world were analyzed in detail to determine their evolutionary relationships. We have constructed a phylogenetic model tree by the neighbour-joining method using nucleotide sequences of 15 haemagglutinin genes, including those of five viruses determined in the present study. This gene tree revealed the existence of two major evolutionary pathways during a twenty five-year period between 1963 to 1988, and each pathway appeared to consist of two distinct lineages of haemagglutinin genes. Furthermore, our analysis of nucleotide sequences showed that two distinct lineages of equine H3N8 viruses were involved in an equine influenza outbreak during the period of December 1971-January 1972 in Japan. The number of nucleotide changes between strains was proportional to the length of time (in years) between their isolation except for three of the HA genes. However, there are three exceptional strains isolated in 1971, 1987, and 1988, respectively. The haemagglutinin gene in these strains showed a small number of nucleotide substitutions after they branched off around 1963, suggesting an example of frozen replication. Although the estimated rate (0.0094/site/year) of synonymous (silent) substitutions of the haemagglutinin gene of equine H3N8 viruses was nearly the same as that of human H 1 and H 3 haemagglutinin genes, the rate of nonsynonymous (amino-acid changing) substitutions of the former equine virus gene was estimated to be 0.00041/site/year - that is about 5 times lower than that estimated for the human H 3 haemagglutinin gene. The present study is the first demonstration that multiple evolutionary lineages of equine H3N8 influenza virus circulated since 1963. [ABSTRACT FROM AUTHOR]

Two influenza A viruses were isolated from pigs in Thailand in January 1988 during the early febrile stage of an influenza-like illness. The isolates contained hemagglutinin and neuraminidase antigens related to those of swine H1N1 influenza virus. This result based on the virus isolation is compatible with the epizootiological evidence that, unlike the human influenza with peak activity in summer (May-July), swine influenza virus is prevalent in the winter season (November-January) in Thailand. The proportion of sera with hemagglutination-inhibiting antibody was higher to A/NJ/8/76 than to A/sw/Iowa/15/30. Likewise, hemagglutination-inhibition tests with monoclonal antibodies indicated that hemagglutinin antigen of the isolates was very similar to that of A/NJ/8/76 virus. In agreement with the serological survey and antigenic characteristic, genetic relatedness between the isolates from Thailand and A/NJ/8/76 virus was also demonstrated by the oligonucleotide mapping of RNA, suggesting that they may be of the same origin. [ABSTRACT FROM AUTHOR]

Growth characteristics of a wide range of influenza A viruses from different mammals and bird species were examined in an established line of canine kidney (MDCK) cells at an ordinary (37°C) and a high temperature (42°C). Although all viruses employed in the present study possessed a capability of replicating at 37°C, virus growth at 42°C showed considerable variation and reflected differences in the natural hosts of the isolates. All reference strains and isolates from bird species grew well in the MDCK cells maintained at 42°C, but human viruses did not, showing an asymmetrical growth behavior. In contrast to this, growth of swine and equine viruses showed growth characteristics intermediate between human and avian viruses. Of the two swine viruses examined, replication of one strain occured equally well at both temperatures and another failed to grow at 42°C. Similarly, two of the three equine viruses tested belonging to H3N8 antigenic subtypes grew at 42°C. However, the results obtained from comparison of plaque sizes and growth curves indicated that the replication of the above swine and equine viruses was restricted under a stringent temperature when compared to avian viruses. The detailed analysis of cloned viruses revealed that some of the swine and equine viruses contained two variants which are readily distinguished by growth behavior at 42°C. Genome analysis of parental and virus clones by oligonucleotide mapping and migration profiles of RNA segments did not detect any differences among the above variants exhibiting the asymmetrical growth characteristics at 42°C. [ABSTRACT FROM AUTHOR]

A recombinant (H1N2, formerly Hsw1N2), 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 (22). 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°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. [ABSTRACT FROM AUTHOR]

The hemagglutinin-neuraminidase (HN) subunits of NDV and NDV-like isolates were analyzed antigenically by monoclonal antibodies to the HN of Miyadera and Taka viruses. In immuno-double-diffusion (IDD) tests, all NDVs examined gave clear lines of precipitation with some of the potent monoclonal antibodies, but it was difficult to determine with certainty the immunological properties of HN subunits due to a rare disagreement with the results obtained in other immunological tests. Monoclonal antibodies used in the tests were found to show different immunological reactivities with the viruses. Monoclonal antibodies belonging to the 1st group (1/29) inhibited the hemagglutinating (HA) activity of all strains but not the neuraminidase (NA) activity. The second monoclonal antibody (5/205) inhibited both the HA and NA activities of the restrictive NDV strains, indicating antigenic changes in HN molecules. However, the inhibitory activity of this monoclone to neuraminidase appeared to be greatly diminished when neuraminyl lactose was used as substrate. Although the 3rd type of monoclonal antibody (5/220) showed HI activity against several strains, this antibody did not inhibit NA activity of any viruses. The remaining monoclone to the HN of Taka virus inhibited the HA activity of all reference strains of NDV and many NDV-like isolates but did not affect NA activity. Two inhibitory activities of four monoclonal antibodies against different viruses, HI and hemolysis-inhibition, were not always consistent with inhibition of virus growth. HI and NI tests with the above four monoclonal antibodies showed that the strains tested fell into five antigenic groups according to their reaction patterns with mouse hybridoma antibodies. [ABSTRACT FROM AUTHOR]

Specific antisera for the isolated HN proteins of eight reference strains of avian paramyxoviruses could be prepared in guinea pigs by intraperitoneal injection of guinea pig red blood cells (GRBC) coated with purified HN proteins. In the hemagglutination inhibition (HI) tests, all reference strains reacted strongly with each homologous antiserum to the isolated HN showing that a low level of cross-reactivity among the reference strains was greatly diminished by using specific antisera. Immuno-double-diffusion (IDD) tests showed that all antisera except those to turkey/Wisconsin/68 and duck/Hong Kong/D3/75 gave single well-defined lines only with the homologous viruses. The remaining two antisera developed a single definite precipitin line together with weak lines with homologous virus. Two isolates in Japan were clearly identified in HI and IDD tests with specific antisera to the HN subunits of the reference strains suggesting that the antisera were useful for identification of avian paramyxovirus isolates. Two isolates in Japan, H-70 from a munia-bird and Y-7 from a duck were found to have HN proteins related closely to those of finch/N. Ireland/Bangor/73 and duck/Hong Kong/199/77, respectively. [ABSTRACT FROM AUTHOR]

The characteristics of an avian influenza virus were compared in detail with those of human Asian (H2N2) influenza viruses. Antigenic analysis by different antisera against H2N2 viruses and monoclonal antibodies to both the hemagglutinin and neuraminidase antigens showed that an avian isolate, A/duck/München/9/79 contained hemagglutinin and neuraminidase subunits closely related to those of the early human H2N2 viruses which had been prevalent in 1957. However, this avian virus gave low HI titers with absorbed and non-absorbed antisera to different human H2N2 viruses isolated in 1957. Like human Q phase variant, such as A/RI/5/57 (H2N2), hemagglutination of the above avian strain was not inhibited by the purified non-specific γ-inhibitor from guinea pig serum. Growth behavior at restrictive temperature (42° C) clearly differentiate the avian H2N2 virus from human influenza viruses, showing that the former virus grew well in MDCK cells at 42° C but not the latters. Genomic analysis of these viruses revealed that the oligonucleotide map of H2N2 virus isolated from a duck was quite different from those of human H2N2 viruses from 1957 to 1967. The oligonucleotide mapping also indicated that different H2N2 influenza virus variants had co-circulated in humans in 1957. [ABSTRACT FROM AUTHOR]

A total of 18 hemagglutinating agents were isolated from 14 of 278 migrating feral ducks in Hokkaido during the surveillance studies conducted from 1978 to 1981. Seven of the 18 isolates belonged to paramyxovirus and the rest to influenza A virus. Five isolates of paramyxovirus reacted specifically with antiserum to duck/HK/199/77 and 7 isolates of influenza A virus possessed the antigenic configuration of H10N3. Three of the isolates possessed an hemagglutinin that has no antigenic relation to any of the 26 known strains of avain, swine, equine and human influenza A viruses. [ABSTRACT FROM AUTHOR]

Panosialin is a new type of metabolite, inhibiting sialidases of myxoand paramyxoviruses and various other enzymes under certain conditions. It also inactivated both hemagglutinating activity and infectivity of influenza virus. The inactivating activity of panosialin was more potent than that of sodium dodecyl sulfate or sodium dodecylbenzene sulfonate. Treatment with panosialin causes destruction of influenza virus particles to smaller particles of about 7 to 10 nm in diameter which can be shown by electron microscopy and by the lower sedimentation rate in ultracentrifugation. Panosialin apparently caused disruption of influenza virus particles. The effect of panosialin was reversed by the pretreatment with bovine albumin. [ABSTRACT FROM AUTHOR]

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