Comparison of the three large polymerase proteins of influenza A, B, and C viruses

The three large RNA segments of influenza C virus C/JJ/50 were cloned and sequenced, and the deduced amino acid sequences were compared with those of the polymerase (P) proteins of influenza A and B viruses. The coding strategy of the C virus RNA segments is the same as that for the large A and B virus segments as one long open reading frame is present in each segment. RNA segment 1 of influenza C virus encodes the equivalent of the PB2 protein; it has an approximate 25% sequence identity with the corresponding (cap binding) influenza A and B virus PB2 proteins. The PB1 protein of influenza C virus, coded for by segment 2, has an approximate 40% sequence identity with the corresponding proteins of influenza A and B viruses including the Asp-Asp sequence motif found in many RNA polymerase molecules. The PB1 polymerase is thus the most highly conserved protein among the influenza A, B, and C viruses. Although the protein coded for by RNA 3 of influenza C virus shows an approximate 25% sequence identity with the acid polymerase (PA) proteins of the A and B viruses, its sequence does not display any acid charge features at neutral pH. This protein is thus referred to as the P3 (rather than the PA) protein of influenza C virus.     

Antigenic determinants of human influenza viruses among the influenza viruses isolated from animals

Comparative studies of the antigenic properties of hemagglutinin (HA) of animal and human viruses revealed both similarities between them and complete differences in the composition of antigenic determinants. Avian influenza viruses A/chicken/Kamchatka/12/71, A/pintail/Primorie/730/76, and A/bat/Alma-Ata/73/77 were completely identical with human strains of influenza virus. Influenza A/horse/Miami/63 contains one antigenic determinant H3.1.HA of A/tern/Turkmenia/18/73 (Hav7) viruses has a peculiar set of antigens. Apart from two antigenic determinants H3.1 and H3.3 inherent in human virus strains, HA of A/tern/Turkmenia/18/73 virus contains an antigenic determinant the population of antibodies to which shows no relation to HA of subtypes Hav2-Hav9.     

Antigenic properties of the M1 proteins of influenza type B viruses and the detection of a common antigenic determinant of the M1 proteins of type A and B viruses

Antigenic properties of M1 proteins of influenza B viruses were studied by indirect EIA using monospecific antisera to M1 protein obtained by immunization of rabbits and mice. Antisera with highest titres were obtained by immunization of mice with polyelectrolyte-conjugated M1 protein. Antigenic heterogeneity of M1 proteins of influenza B viruses was demonstrated. M1 proteins of B/Lee/40 and B/USSR/03/84 viruses reacted with B/Hong Kong/72 antiserum to one-fourth of the homologous titre. Cross-experiments using M1 proteins recovered from influenza type A and B viruses and antisera to them revealed heterologous reactions suggesting a common antigenic determinant in A and B M1 proteins. Treatment of the antisera with M1 protein of type A resulted in a decline of antibody titres to M1 proteins of type B. The two available monoclones to M1 protein of type A reacted only with M1 A but not with M1 B.     

Antigenic drift and variability of influenza viruses

Annual influenza epidemics are caused by rapid evolution of the viral genome. Continuous and extensive antigenic variation has been shown for hemagglutinin (HA), the principal immunizing antigen of the virus. Monitoring of the antigenicity of circulating influenza viruses is necessary for selection of the most suitable vaccine strains. In this study, characterization of influenza A/H3N2 and influenza B viruses recently circulating in Germany was performed by molecular and antigenic analysis. Sequencing and phylogenetic analysis of the HA1 gene revealed that two distinct groups of H3N2 viruses co-circulated during 1997/1998. The majority of isolates clustered with the new drift variant A/Sydney/5/97, as was also shown by antigenic characterization. A noteworthy genetic drift of H3N2 viruses was evident during the winter 1998/1999. However, serological characterization using hemagglutinin inhibition tests did not result in detection of viruses belonging to different groups as confirmed by molecular analysis. Influenza B viruses isolated during 1996/1997 were antigenically closely related to the prototype vaccine strains B/Beijing/184/93 or B/Harbin/7/94. Molecular analysis demonstrated that our German 1996/1997 isolates differed by nine amino acids from B/Harbin/7/94 and represented a group of viruses that was completely different from the Harbin strain. Retrospective studies revealed the circulation of B/Yamanshi/166/98-like viruses in Germany already during the 1996/1997 season. Our results suggest that molecular analysis of the HA gene is important to complement the antigenic characterization for a better selection of appropriate vaccine strains.     

Detection and identification of human influenza viruses by the polymerase chain reaction

A series of oligonucleotide primers are described which hybridize to conserved regions of influenza virus cDNA and prime DNA synthesis in Taq polymerase catalyzed amplification reactions (PCR). Primers were designed to hybridize as nested pairs and, following a two-step amplification, produce uniquely sized DNA fragments diagnostic for viral type and subtype. Influenza A and B matrix-protein genes and the influenza C haemagglutinin gene were targets for the type-specific primers. Subtype-specific primers targeted conserved sequences within the three haemagglutinin or two neuraminidase subtypes of different human influenza isolates. The utility of this method was demonstrated using computer search methods and by accurately amplifying DNA from a variety of influenza A, B, and C strains. Type-specific primer sets showed a broad type specificity and amplified DNA from viral strains of unknown sequence. Restriction mapping and DNA sequencing showed that fragments amplified in this manner derived from the input template, confirming the accuracy of the method and demonstrating how PCR can be used to quickly derive sufficient sequence information for analysis of viral relatedness. Subtyping primers were able to distinguish accurately between the three haemagglutinin (H1, H2, H3) and two neuraminidase (N1, N2) alleles of human influenza A isolates. Again DNA was amplified from viruses of unknown sequence confirming that most of these primer sets may prove useful as broad range subtyping reagents. In order to simplify the work associated with analysis of many samples, we have also devised a rapid method for the isolation of viral RNA and synthesis of cDNA. Using this 'mini-prep' technique, it is possible to detect, amplify, and identify picogram quantities of influenza virus in a single day, confirming that PCR provides a useful alternative to existing methods of influenza detection.     

Categorization of nucleoproteins and matrix proteins from type A influenza viruses by peptide mapping

Relationships between the nucleoprotein (NP) and matrix protein (M) of 16 human and 3 lower vertebrate strains of type A influenza virus were investigated by two-dimensional mapping of [³⁵S]methionine peptides. By regarding certain common groups of peptides as taxonomic criteria, all NP proteins could be classified as belonging to one of two basic patterns. Differences existed between maps of either category but these have not been investigated in detail. The chymotryptic-tryptic peptide maps of the M proteins were too similar overall to warrant dividing them into subgroups but differences between individual strains were clearly apparent. Both patterns of NP protein occurred within a subtype. The change from one NP pattern to another, and back again, within a subtype is consistent with the hypothesis that genetic assortment of NP genes occurs during the reign of a particular subtype. This and other implications are discussed.     

Reproduction of human and animal influenza viruses in human nasal polyp organ cultures

Human influenza virus strains were easily grown and passaged in human nasal polyp organ cultures causing marked damage of the epithelium. Unlike to human strains, the animal influenza virus strain could be propagated for no longer than 2 or 3 passages and even the 1st passage failed to cause significant morphological changes of the epithelium cells.     

Antigenic determinant composition of the hemagglutinins of influenza type B viruses isolated in 1940-1975

Three antigenic determinants in influenza type B viruses hemagglutinin characterising similarities and differences between these viruses were found using monoreceptor sera. The viruses contained the following antigenic determinants: B/Lee/40 B1, B2, B3; B/Cri/49 B1, B2, B4; BM8/52 B1, B5, B6; B/60, 59 B1, B5, B7; B/Victoria/70 B1, B5, B8, and B/Hong Kong/7/75 B1, B3, B8.     

Antigenic relationship between the surface antigens of avian and Equine influenza viruses

Influenza virus Equine 1 (A/equine/Prague/56) has a hemagglutinin which is antigenically related to the hemagglutinin of fowl plague virus strain Rostock (FPV) and a neuraminidase which cross-reacts with the enzyme of virus N (A/chick/Germany/49). After a single injection of chickens with Equine 1 virus no hemagglutination inhibiting (HI) and neutralizing antibodies against FPV can be demonstrated, although the birds are fully protected against a lethal dose of FPV. HI and neutralizing antibodies against FPV appear after a second injection of Equine 1 virus several weeks after the first one. Liberation of newly sunthesized FPV from the host cell is ingibited by antibodies cross-reacting with any antigen of virus surface.     

Antigenic structure of the hemagglutinin of H9N2 influenza viruses

The hemagglutinins (HAs) of H9 influenza viruses isolated from birds and mammals of different species were antigenically and genetically analyzed. Antigenic variants were selected from A/swine/Hong Kong/10/98 (H9N2) and A/duck/Hokkaido/13/00 (H9N2) in the presence of monoclonal antibodies (MAbs). Based on the reactivity patterns of these mutants with a panel of MAbs, at least five non-overlapping antigenic sites were defined using eight MAbs which recognized seven distinct epitopes on the H9 HA molecule. Based on the reactivity patterns with the panel of monoclonal antibodies, 21 H9N2 virus strains isolated from birds and mammals were divided into 7 antigenically distinct groups. The present findings indicate that it is important to monitor the antigenic variation in H9 influenza viruses. The panel of MAbs in the present study, thus, should be useful for detailed antigenic analysis of the H9 HAs for epidemiological studies, the selection of vaccine strains, and diagnosis.     

Antigenic composition of the hemagglutinins of influenza viruses H2N2

A comparative immunological analysis of the antigenic structure of hemagglutinins of influenza H2N2 viruses isolated in 1957-1967 was carried out by the method of specific adsorption of highly active antiviral rabbit sera. The hemagglutinins of the viruses under study were shown to contain at least three antigenic determinants changing in the course of evolution, one of them shared by all the viruses of the H2N2 subtype remaining antigenically stable since 1957 until 1967.