Differences between influenza virus receptors on target cells of duck and chicken and receptor specificity of the 1997 H5N1 chicken and human influenza viruses from Hong Kong.

To study whether influenza virus receptors in chickens differ from those in other species, we compared the binding of lectins and influenza viruses with known receptor specificity to cell membranes and gangliosides from epithelial tissues of ducks, chickens, and African green monkeys. We found that chicken cells contained Neu5Ac alpha(2-6)Gal-terminated receptors recognized by Sambucus nigra lectin and by human viruses. This finding explains how some recent H9N2 viruses replicate in chickens despite their human virus-like receptor specificity. Duck virus bound to gangliosides with short sugar chains that were abundant in duck intestine. Human and chicken viruses did not bind to these gangliosides and bound more strongly than duck virus to gangliosides with long sugar chains that were found in chicken intestinal and monkey lung tissues. Chicken and duck viruses also differed by their ability to recognize the structure of the third sugar moiety in Sia2-3Gal-terminated receptors. Chicken viruses preferentially bound to Neu5Ac alpha(2-3)Gal beta(1-4)GlcNAc-containing synthetic sialylglycopolymer, whereas duck viruses displayed a higher affinity for Neu5Ac alpha(2-3)Gal beta(1-3)GalNAc-containing polymer. Our data indicate that sialyloligosaccharide receptors in different avian species are not identical and provide a potential explanation for the differences between the hemagglutinin and neuraminidase proteins of duck and chicken viruses.

Differences between HA receptor-binding sites of avian influenza viruses isolated from Laridae and Anatidae.

A comparative study of the hemagglutinin (HA) receptor binding site (RBS) of a number of H13 influenza viruses isolated from Laridae family of birds (gulls) and other influenza viruses obtained from the Anatidae family (ducks) was conducted. The affinity of all viruses to alpha N-acetylneuraminic acid (Neu5Ac alpha), 3'sialyllactose (3'SL), and sialylglycopolymers bearing 3'-sialyl(N-acetyllactosamine) (3'SLN-PAA), [Neu5Ac alpha(2-3)Gal beta(1-4)][-Fuc alpha(1-3)]GlcNAc beta (SLe(x)-PAA), and [Neu5Ac alpha(2-3)Gal beta(1-3)][-Fuc alpha(1-4)]GlcNAc beta (SLe(a)-PAA), was determined. The last three polymer glycoconjugates were synthesized for determining the contribution of carbohydrate chains after the galactose link to the binding with the receptor. The difference in affinity between 3'SL and Neu5Ac alpha in all studied H13 viruses is small, which indicates a less significant role of the galactose moiety in the binding to the receptor. The results of virus binding with polymer sialylglycoconjugates indicates that the method of linking, the third monosaccharide moiety, and the presence of an extra fucose substitute in this moiety may influence the binding considerably. For viruses isolated from ducks, the suitable polymer is SLe(a)-PAA (i.e., a 1-3 linkage between galactose and glucosamine is optimal). This finding is in accord with the data that H13 viruses isolated from the gulls differ based on their ability to interact with polymer sialylglycoconjugates. The affinity to all three polymers is uniform, and the presence of GlcNAc-linked fucose does not prevent the binding. A comparative analysis of six sequenced HA H13 viruses and other subtype viruses showed presence of substantial differences in the composition of amino acids of this region in H13 viruses.

Extracellular truncated influenza virus nucleoprotein.

In the culture medium of MDCK cells infected with influenza A/Duck/Ukraine/1/63(H3N8) virus two kinds of virus nucleoprotein (NP) are detected: full-length 56 kDa NP and truncated 53 kDa NP. However, in infected cells 53 kDa NP may be detected only at short pulse and after 10 min chase it becomes nondetectable. The extracellular truncated 53 kDa NP is detected in free RNP, and not in the virions. Both extracellular free 53 and 56 kDa NP in the virions are completely oligomerized. Several data argue against the possibility of extracellular 53 kDa NP formation being a result of extracellular 56 kDa NP proteolytic degradation. Thus, the accumulation of extracellular 53 kDa NP takes place only in the course of infection, and the amount of 53 kDa NP is not increased during prolonged storage of cell-free culture medium at +37 degrees C. Moreover, all extracellular 56 kDa NP of A/Duck/Ukraine/1/63 influenza virus is present in the oligomeric form, and the latter, in contrast to the mononeric form, is highly resistant to proteases. The possibility is discussed that in the course of A/Duck/Ukraine/1/63 (H3N8) influenza virus infection a fraction of the synthesized 56 kDa monomeric NP undergoes the proteolytic cleavage in the infected cells before oligomerization and forms the 53 kDa NP. This 53 kDa NP is then oligomerized, enters the RNP and is quickly secreted from the cells.

Avian and human influenza a virus strains possess different intracellular nucleoprotein oligomerization efficiency.

We have previously shown (Prokudina-Kantorovich EN and Semenova NP, Virology 223, 51-56, 1996) that the nucleoprotein (NP) of influenza A virus forms in infected cells oligomers which in the presence of SDS and 2-mercaptoethanol (ME) as reducing agent are stable at room temperature (RT) and dissociate at 100 degrees C. Here we report that the efficiency of intracellular NP oligomerization depends on the host origin of influenza A virus strain. Thus, in the cells infected with avian influenza A virus strains the viral NP was almost completely oligomerized and only traces of monomeric NP were detected by polyacrylamide gel electrophoresis (PAGE) in unboiled samples. However, in the cells infected with human influenza A virus strains, besides oligomeric NP also a significant amount of non-oligomerized monomeric NP was detected in unboiled samples. In purified virions of avian and human strains the same difference in NP monomers/oligomers ratio was detected as in the infected cells. A reassortant having all internal protein genes from a human strain and the glycoprotein genes from an avian strain revealed the same intracellular pattern of NP monomers/oligomers ratio as its parental human virus. These findings suggest that the type of NP oligomerization is controlled by the NP gene. The possible connection between the accumulation of protease-sensitive monomeric NP in cells infected with a human influenza strain and the parallel accumulation of cleaved NP in these cells is discussed.

Cross-protection and reassortment studies with avian H2 influenza viruses.

In order to assess the degree of immune cross-protection among avian H2 influenza virus strains, mice were immunised with beta-propiolactone-inactivated virus preparations and infected intranasally with mouse-adapted variant of A/Black Duck/New Jersey/1580/78 (H2N3) strain. The experiments with 11 avian H2 strains revealed that both Eurasian and American H2 avian influenza viruses exhibit either high or moderate degree of cross-protection. The grouping of the strains in accordance with their cross-protection efficiency does not coincide with H2 phylogenetic branches. Several reassortant clones were obtained with the use of A/Pintail Duck/Primorie/695/76 (H2N3) strain and high-yield X-67 reassortant as parent viruses, among them a high-yield H2N3 reassortant. Taking into account the data on cross-protection among avian H2 strains, the high-yield H2N3 reassortant may be regarded as a prototype strain to be used for the preparation of killed vaccines in the case of a new appearance of avian H2 haemagglutinin in circulation in humans.

Precursor genes of future pandemic influenza viruses are perpetuated in ducks nesting in Siberia.

Influenza A viruses of different subtypes were isolated from fecal samples of ducks in their nesting areas in Siberia in summer from 1996 to 1998. Phylogenetic analysis of the NP genes of the isolates in Siberia and those in Hokkaido, Japan on their flyway of migration from Siberia to the south in autumn revealed that they belong to the Eurasian lineage of avian influenza viruses. It is noted that the genes of the isolates in Siberia are closely related to those of H5N1 influenza virus strains isolated from chickens and humans in Hong Kong in 1997 as well as to those of isolates from domestic birds in southern China. The results indicate that influenza viruses perpetuated in ducks nesting in Siberia should have contributed genes in the emergence of the H5N1 virus in Hong Kong. Vaccine prepared from avirulent A/duck/Hokkaido/4/96 (H5N3) influenza virus was potent enough to protect mice from challenge with lethal dose of the pathogenic H5N1 virus [19]. Intensive surveillance study of aquatic birds especially in Siberia is, therefore, stressed to provide information on the future pandemic influenza virus strains and for vaccine preparation.

Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site.

Avian influenza virus strains representing most hemagglutinin (HA) subtypes were compared with human influenza A (H1N1,H3N2) and B virus isolates, including those with no history of passaging in embryonated hen's eggs, for their ability to bind free N-acetylneuraminic acid (Neu5Ac) and sialylollgosaccharides in a competitive binding assay and to attach to gangliosides in a solid-phase adsorption assay. The avian viruses, irrespective of their HA subtype, showed a higher affinity for sialyl-3-lactose and the other Neu5Ac2-3Gal-terminated oligosaccharides and a lower affinity for sialyl-6-lactose than for free Neu5Ac, indicative of specific interactions between the HA and the 3-linked Gal and poor accommodation of 6-linked Gal in the avian receptor-binding site (RBS). Human H1 and H3 strains, by contrast, were unable to bind to 3-linked Gal, interacting instead with the asialic portion of sialyl-6-(N-acetyllactosamine). Different parts of this moiety were recognized by H3 and H1 subtype viruses (Gal and GlcNAc, respectively). Comparison of the HA amino acid sequences revealed that residues in positions. 138, 190, 194, 225, 226, and 228 are conserved in the avian RBS, while the human HAs harbor substitutions at these positions. A characteristic feature of avian viruses was their binding to Neu5Ac2-3Gal-containing gangliosides. This property of avian precursor viruses was preserved in early human H3 isolates, but was gradually lost with further circulation of the H3 HA in humans. Consequently, later human H3 isolates, as well as H1 and type B human strains, were unable to bind to short Neu5Ac2-3Gal-terminated gangliosides, an incompatibility that correlated with higher glycosylation of the HA globular head of human viruses. Our results suggest that the RBS is highly conserved among HA subtypes of avian influenza virus, while that of human viruses displays distinctive genotypic and phenotypic variability.

Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6'-sialyl(N-acetyllactosamine).

Synthetic sialylglycoconjugates bearing 3'-sialyllactose, 6'-sialyllactose, or 6'-sialyl(N-acetyllactosamine) moieties attached to the polyacrylic acid carrier (P-3-SL, P-6-SL, and P-6-SLN, respectively) were prepared and tested for their ability to bind to influenza virus isolates from different hosts in a competitive solid phase assay. The virus panel included egg-grown avian and porcine strains, as well as human viruses isolated and propagated solely in mammalian (MDCK) cells and their egg-adapted variants. A clear correlation was observed between the pattern of virus binding of two glycopolymers, P-3-SL and P-6-SLN, and the host species from which the virus was derived. Avian isolates displayed a high binding affinity for P-3-SL and a two to three orders of magnitude lower affinity for P-6-SLN. By contrast, all non-egg-adapted human A and B viruses bound P-6-SLN strongly but did not bind P-3-SL. Unlike the "authentic" human strains, their egg-adapted counterparts acquired an ability to bind P-3-SL, indicative of a shift in the receptor-binding phenotype toward the recognition of Neu5Ac2-3Gal-terminated sugar sequences. Among the porcine viruses and human isolates with porcine hemagglutinin, few displayed an avian-like binding phenotype, while others differed from both avian and human strains by a reduced ability to discriminate between P-3-SL and P-6-SLN. Our data show that sialylglycopolymers may become a useful tool in studies on molecular mechanisms of interspecies transfer, tissue specificity, and other structure-function relationships of the influenza virus hemagglutinin.

Phenotypic expression of HA-NA combinations in human-avian influenza A virus reassortants.

Human-avian and human-mammalian influenza A virus reassortant clones with the neuraminidase (NA) gene of the A/USSR/90/77 (H1N1) strain and hemagglutinin (HA) genes of H3, H4 and H13 subtypes had been shown in an earlier publication to produce low HA yields in the embryonated chicken eggs. The low HA titers had been shown to be due, at least in part, to the formation of virion clusters at 4 degrees C; the clustering was removed by the treatment with bacterial neuraminidase [Rudneva et al., Arch. Virol (1993) 133: 437-450]. By serial passages of the reassortants in chick embryos non-aggregating variants were selected: the variants produced HA titers of the same order as A/USSR/90/77 parent virus. The assessment of the virus yields by the analysis of the partially purified virus preparations from fixed volumes of the allantoic fluid revealed that actual virion yields of the initial reassortants were lower than the yields of their passaged variants or of the parent viruses. The passaged variant of a reassortant possessing the HA gene of A/Duck/Ukraine/1/63 (H3N2) virus differed from the original (non-passaged) reassortant and from the parent A/Duck/Ukraine/1/63 virus in the reaction with a panel of monoclonal antibodies against H3 hemagglutinin. The data suggest that some HA-NA combinations may lead to an incomplete functional match between HA and NA and to the formation of low-yield reassortants, thus representing a possible limiting factor in the emergence of new HA-NA combinations in natural conditions.

A reassortant H1N1 influenza A virus caused fatal epizootics among camels in Mongolia.

In the autumn of 1979 a severe influenza epizootic started among camels in Mongolia (Lvov et al., 1982; Viprosi Virusol. 27, 401-405.) Between 1980 and 1983 13 independent isolates of H1N1 viruses were obtained from diseased camels, which were virtually indistinguishable from the human A/USSR/90/77 strain by serological means. Two hundred and seventy-one samples of camel sera collected between 1978 and 1983 contained antibodies against the human A/USSR/90/77 isolate. After experimental infection of camels with some of these isolates, the animals developed similar symptoms as those found during natural infection: coughing, bronchitis, fever, discharge from nose and eyes. A genetic sequence analysis revealed that among the eight segments (genes) the PB1, HA, and NA genes were almost identical with allelic genes of the USSR/77 strain, and the PB2, PA, NP, M, and NS genes were almost identical with those of the A/PR/8/34 strain.

Influenza A virus reassortants with surface glycoprotein genes of the avian parent viruses: effects of HA and NA gene combinations on virus aggregation.

A series of 33 human-avian and human-mammalian influenza virus reassortant clones possessing either HA or both HA and NA genes of the avian or mammalian virus was obtained by crosses of A/USSR/90/77 (H1N1) human virus with 5 avian and 1 mammalian influenza virus strains. All of the reassortants possessing NA genes of the H1N1 human parent virus and HA gene of an avian or mammalian parent virus had high values of infectivity/HA activity ratio. Since this feature could result from a limited virion aggregation, several reassortants were analyzed by velocity sucrose gradient centrifugation. In all cases tested, the reassortants of H3N1, H4N1, H10N1 and H13N1 composition were shown to be aggregated, whereas the preparations of the parent H1N1 virus and the reassortants possessing both HA and NA genes from the avian parents were represented mostly by single virions. The aggregates were formed at 4 degrees C and dissociated at 37 degrees C. The dissociation was blocked by an inhibitor of neuraminidase activity (2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid). The dissociation was reversible since the virions reaggregated at 4 degrees C; however, treatment with bacterial neuraminidase led to an irreversible dissociation of the aggregates. The tendency of the reassortants to aggregate correlates with an increased infectivity/HA ratio. No regular decrease in the neuraminidase activity in the virions of reassortants as compared to the parent H1N1 virus was revealed. The most likely explanation of the observed phenomenon seems to be an inefficient removal of sialic acid residues from the avian virus hemagglutinin by the human virus N1 neuraminidase.

Formation of mixed hemagglutinin trimers in the course of double infection with influenza viruses belonging to different subtypes.

Chick embryo primary cultured cells were infected with influenza viruses belonging to H1, H2, H3, H5 or H7 subtypes of hemagglutinin. The cells were subjected to a single or a double infection, labelled with 14C-amino acids from 2 to 6 hours postinfection, lysed with a mixture of ionic and non-ionic detergents, and the lysates were clarified by low-speed centrifugation. The clarified lysates contained 14C-labelled hemagglutinin mostly in the form of 9S trimers, as shown by velocity sedimentation in sucrose gradients with polyacrylamide gel electrophoresis (PAGE) analysis of the gradient fractions. The lysates were immunoprecipitated with antihemagglutinin antibodies specific for one of the co-infecting viruses. The immunoprecipitates were analysed by PAGE. Cells infected separately with each virus and mixed before lysis were used as a control sample in every experiment. In the lysates of cells doubly infected with H2 and H5 influenza viruses the analysis revealed the presence of structures containing HA monomers of both viruses, whereas no such structures were revealed in the lysate of a mixture of separately infected cells. Mixed structures (most likely HA trimers containing monomers of the two co-infecting viruses) were also found in the lysates of cells doubly infected with strains belonging to H1 and H2 subtypes. No such structures were revealed when the cells were co-infected with viruses belonging to H1 and H3 subtypes or H3 and H7 subtypes. The results suggest an extensive formation of mixed HA trimers in the course of double infection with viruses belonging to closely related subtypes, whereas the formation of mixed trimers by more distantly related HA monomers does not occur or is very scarce. The identity of the mixed structures as HA trimers was confirmed by immunoprecipitation experiments with 9S structures.

Effect of beta-decay of radionuclides incorporated into influenza virus RNA and proteins on the infectivity of the virus and antigenicity of its nucleoprotein.

The effect of beta-decay of radionuclides incorporated into influenza virus on the properties of the two closely adjacent structures--RNA and nucleoprotein (NP)--was studied. The long-term storage of 3H-uridine labelled influenza virus was shown to lead to the loss of infectivity. This effect may be explained by lethal intra-molecular modifications of viral RNA, caused by beta-decay of 3H incorporated into the molecule. There was an accompanying decrease of monoclonal antibody (MAB) binding activity, this also being a plausible result of beta-decay. The different rates of inactivation of MAB binding activity of different epitopes of NP of the 3H-labelled virus shown in our studies suggest that there are different types of structural organization or different location of these epitopes in the NP. The effect of 3H-decay on the intracellular RNA of reproducing virus lead to a decrease in virus yield; this may be due to radiation- and transmutation-induced damage of messenger and progeny RNA populations synthesized during the infection. The storage of influenza virus labelled with 14C-aminoacids lead to a decrease in MAB binding activity of the NP that was unaccompanied by a decrease in infectivity. Furthermore, 14C-decay in proteins of reproducing virus had no adverse effect.

Primer informe de aislamiento del virus de la gripe A en aves silvestres en Cuba / First report about isolation of influenza A virus from wild birds in Cuba

Se cazan 100 aves silvestres autóctonas y migratorias en la provincia de Matanzas, Municipio Ciénaga de Zapata. Se obtienen 9 aislamientos para el 9% de positividad. Se informa que de los aislamientos obtenidos, ocho se correspondieron con la fórmula antigénica H3N8, los cuales fueron recombinantes del Complejo Hong Kong; 1 se corresponde con la fórmula antigénica H7N7 similar a la cepa A/Equina/Praga/1/56 (H7Ny) por su neuroaminidasa, y por su hemaglutinina a la cepa A/FPV/Rostock/34 (H7N1)

DNA sequences in influenza virions.

During the propagation of A (H3N2) influenza virus in chick embryos, incorporation of 3H-thymidine into virions takes place, whereas no such incorporation occurs with Newcastle disease virus. Incorporation of 3H-thymidine is a result of DNA synthesis. This virion-associated DNA is present in cores obtained after treatment of virions with bromelain.