Preparation of influenza B virus recombinant strains

The study of antigenic and biologic properties of influenza B epidemic viruses isolated in 1979 and 1983 and laboratory strain B/Lee/40 has revealed some differences in their biologic properties. The most marked changes have been found in the haemagglutinin (HA) and neuraminidase (NA) indicating that influenza B viruses underwent dramatic antigenic drifts during the period in question. The strains obtained by genetic recombination have inherited surface antigens of epidemic influenza B/Singapore/222/79 and B/USSR/100/83 viruses and preserved the HA thermolability inherent to these viruses. They have, however, acquired the marker of reproduction in chick embryos and the immunogenicity from the donor strain B/Lee/40. These recombinants can be, therefore, recommended as vaccine strain candidates.     

Antigenic characteristics of influenza B virus strains isolated in an orphanage during an influenza outbreak in Moscow in the winter of 1988

Examinations of ARD patients in an orphanage for defective children in Moscow during an influenza outbreak in the winter of 1988 yielded 12 influenza virus strains, including 6 influenza B strains and 6 influenza A (H3N2) strains. The antigenic analysis of hemagglutinin of influenza B virus isolates showed that with respect to the B/Leningrad/179/86 strain (an antigenic analogue of B/Ann Arbor/1/86 strain recommended for inclusion into the influenza vaccine for 1987-1988) they could be divided into 2 groups: antigenically close to the B/Leningrad/86 strain (isolate B/712) and markedly differing from it (the remaining isolates). As compared with reference strains of the previous years, all the new virus isolates fell into 4 groups: isolate B/712 antigenically related to B/Hong Kong/73 and B/Leningrad/86 strains; B/722 antigenically close to B/Singapore/222/79 and B/USSR/100/83 strains; B/724, an antigenic analogue to B/USSR/100/83; and the remaining isolates, to some or other extent, differing from various reference strains. This attests to simultaneous circulation of various antigenic variants of influenza B virus during the 1988 winter outbreak of influenza in Moscow. An interesting feature of the B/712/88 isolate consists in its antigenic specificity of hemagglutinin being indistinguishable from that of B/Hong Kong/8/73 strains.     

Isolation of the influenza B virus in MDCK cells

The frequency of influenza B virus isolation from clinical specimens is much higher when a continuous line of dog kidney cells, MDCK, is employed, and not the developing chick embryos. Among 9 influenza B virus strains isolated during the influenza epidemic of 1983-1984 winter, 8 strains were isolated in MDCK cells and only 1 in chick embryos. The influenza B virus isolates were similar to influenza B/Singapore/222/79 virus differing from it in HI titres 2-16-fold.     

Increased sensitivity and reduced specificity of hemagglutination inhibition tests with ether-treated influenza B/Singapore/222/79

Hemagglutination inhibition (HI) tests against whole virus (WV) influenza B/Singapore/222/79 antigen detected prevaccination serum antibody in only 15 (20%) of 50 predominantly elderly volunteers and fourfold or greater titer rises in only three (6%) after they received 1981-1982 trivalent influenza vaccine containing antigens of this virus. HI titers against ether-treated (ET) B/Singapore/222/79 were about eightfold higher than those against WV antigen and were comparable to microneutralization titers against this virus. The ET HI detected prevaccination antibody in 84%, a postvaccination titer rise in 32%, and a final titer of 80 or higher in 66%. Among 51 additional persons with known or presumed influenza B virus infections early in 1982, ET B/Singapore/222/79 was also more sensitive than WV for serodiagnosis (69 versus 49%), but eight persons with both WV and ET B/Singapore/222/79 HI responses also had an HI titer rise to WV A/Brazil/11/78 (H1N1) antigen. Conversely, among 14 college students with febrile, culture-proven influenza A (H1N1) infections early in 1982, 6 (43%) developed HI titer rises to ET B/Singapore/222/79 with no other serological evidence of influenza B virus infection. Moreover, young adult volunteers with mild experimental influenza A (H1N1) infections also exhibited a 17% (3 of 18) incidence of ET B/Singapore/222/79 HI titer rises, versus none in matched, uninfected volunteers. These data indicate that ET B/Singapore/222/79 virus has increased sensitivity but reduced specificity compared to WV as an HI antigen and that caution is needed in interpretation of a single HI test for serodiagnosis, whether with WV or ET antigen.     

Influenza B virus genome: complete nucleotide sequence of the influenza B/lee/40 virus genome RNA segment 5 encoding the nucleoprotein and comparison with the B/Singapore/222/79 nucleoprotein

The complete nucleotide sequence of a cloned full-length DNA copy of genome RNA segment 5 of influenza B/Lee/40 virus has been determined. The genome segment is 1841 nucleotides in length and is capable of coding for a nucleoprotein (NP) of 560 amino acids. Comparison with the only other known sequence of an influenza B virus nucleoprotein gene (B/Singapore/222/79) indicates striking homology. Only 113 nucleotide substitutions are present between the two strains in their protein coding region and these lead to only 22 amino acid substitutions between nucleoproteins of identical polypeptide chain length. Assuming a common lineage, this reflects a calculated rate of amino acid sequence divergence of 0.1% per year. Like its influenza A virus counterpart, the influenza B/Lee/40 nucleoprotein is a basic protein with a relatively even distribution of its charged residues. The remarkable conservation of nucleoprotein primary structure over a 39-year period probably reflects both selection for performance of specific functions and protection from antigenic selection by the host immune system.     

Differences in the electrophoretic migration rates of polypeptides and RNAs of recent isolates of influenza B viruses

Summary The electrophoretic migration rates of structural and non-structural poly-peptides of 38 influenza B viruses isolated in epidemics in 1978–1980 and antigenically closely related to B/Singapore/222/79 virus were compared using high resolution SDS polyacrylamide gels. Thirty of the viruses could be distinguished from the prototype B/Singapore/222/79 virus by electrophoretic migration rate differences in HA, 17 by differences in NP and 27 by differences in mobility of the NS1 polypeptide. Mobility differences of NP, NS1 and HA polypeptides was noted in influenza B viruses isolated in the UK in the same year. In addition, electrophoretic mobility of³²P labelled virus RNAs varied for certain UK isolates and indicated heterogeneity in genes 2, 3, 4 and 8 coding for polymerase proteins 2 and 1, nucleoprotein (NP) and non-structural protein (NS1) respectively.With 3 Figures     

A comparative analysis of epidemic strains of the influenza A virus (H1N1), isolated in the USSR and Czechoslovakia in 1986

A comparative study of antigenic and molecular-biological properties of epidemic influenza virus strains isolated in the USSR and CSSR in the autumn-winter, 1986-1987 was carried out and showed that the epidemics in both countries were due to influenza A viruses of H1N1 subtype, antigenically related to the reference A/Taiwan/1/86 and A/Singapore/6/86 strains, which was confirmed by electrophoretic studies. The strains were found to vary in sensitivity to heating and to the inhibitors.     

Immunogenicity of a single dose of trivalent influenza vaccine including A/Philippines (H3N2): results of a field trial

During 1982, a new A(H3N2) influenza virus subtype, A/Philippines/2/82, was identified, and this strain was combined with previous A(H1N1) and B influenza virus strains in the trivalent inactivated vaccine recommended for the 1983-1984 influenza season. Prior to the widescale use of this vaccine in Israel, a group of 106 young male soldiers was vaccinated under controlled conditions. Before vaccination, antibody titers greater than or equal to 1:40 were found in 14.1% against A/Philippines (H3N2), 18.1% against A/England/333/80 (H1N1), and 13.3% against B/Singapore/222/79. Two weeks following vaccination, 78.9% of the vaccinees for whom repeated blood samples were available, had antibody titers in this range for A/Philippines (H3N2), 92.9% for A/England (H1N1), and 80.0% for B/Singapore. The vaccine was only mildly reactogenic, and there were no cases of absence from work following vaccination. Thus the antibody response of young subjects to a single dose of a vaccine containing a new A(H3N2) subtype was found to be satisfactory, and the side effects experienced were minimal.     

Immunofluorescence test with antigen-loaded erythrocytes: Detection of influenza virus specific IgG,IgA, and IgM antibodies

An immunofluorescence test (IFT) for the detection of influenza virus antibodies was established to supplement the standard serological diagnostical complement fixation test (CFT). Current strains (A/Philippines/2/ 82, A/Brazil/11/78, B/Singapore/222/79) were loaded on formalinized chicken erythrocytes. In contrast to CFT, we can distinguish specific immunoglobulin classes against influenza virus. Unlike CFT, IFT is subtype-specific. A recent infection can be distinguished from a past infection by the differentiation of specific immunoglobulin classes. Anticomplementary factors and hemolytic sera do not influence the result of the IFT. IFT does not require cell cultures and is easy to read.     

Influenza viruses which preconditioned the epidemic rise in Russia in 2002-2003. A resumed circulation of influenza viruses similar to V/Victoria/2/87

According to research, the epidemic rise of influenza was preconditioned, during 2002-2003, in Russia by the circulation of influenza A(H1N1), A(H3N2) and B viruses. The Center of Influenza Ecology and Epidemiology undertook a study of 178 epidemic strains: 41 strains A(H1N1), 116 strains A(H3N2) and 21 strains of influenza B were among them. All strains were isolated in the MDCK cell culture. A simultaneous isolation in embryonated eggs as well as changing of the isolation system from MDCK to embryonated eggs were found to be effective only for influenza A(H1N1) viruses. According to the antigenic analysis, all A(H1N1) viruses were variants of the etalon A/New Caledonia/20/99. The A(H3N2) viral strains' population was heterogeneous by its antigenic properties: among its isolates, there were variants similar to the etalons of A/Moscow/10/99 and of A/Panama/200/99 as well as strains, which weakly reacted with sera of both above etalons; possibly the latter were close to the etalon of A/Fujian/411/02. All epidemic strains of influenza B virus belonged, according to the antigenic properties of hemagglutinin, to the virus group of B/Victoria/2/87-like and were antigenic variants of the etalon of B/Hong Kong/22/01. This confirmed that influenza B viruses with the antigenic hemagglutinin structure of the virus group of B/Victoria/2/87-like, which were not present in Russia for more than 10 years, re-entered the active circulation. An analysis of antigenic properties of neuraminidases (NA) of the mentioned epidemic strains showed their different degrees of relationship with the NA etalons of both evolutionary groups, i.e. B/Victoria/2/87 and B/Yamagata/16/88-like. A study of paired sera obtained from patients showed a growth of antibodies to the etalons of influenza A(H1N1), A(H3N2) and B viruses of the season in question, which confirmed the virology data.     

Information of the Center for Ecology and Epidemiology of Influenza, D. I. Ivanovsky Research Institute of Virology, Russian Academy of Medical Sciences, on the results of the 2009-2010 influenza and acute respiratory viral infection epidemic season (at week 40 of 2009 to week 22 of 2010) in the world and Russia

The paper describes the specific features of the 2009-2010 epidemic season in Russia and the world, which are due to the wide spread of a new pandemic strain of influenza A(H1N1)v virus. There is an unusual early upsurge in the incidence of influenza and acute respiratory viral infection (ARVI) (in October-November 2009) with its peak at weeks 45 to 48 of the year with a succeeding reduction to the seasonal values by its end. The circulation of influenza B virus strains was recorded in February-April 2010, which was responsible for the higher epidemic thresholds of morbidity in a number of Russia's regions. A study of the antigenic properties of the strains defined their relationship to the reference strains A/California/07/2009 (H1N1)v and B/Brisbene/60/2008. There were strains with amino acid substitutions at position 222 of hemagglutinin in the population of pandemic influenza A(H1N1)v virus. The strains of the new pandemic influenza A(H1N1)v virus were resistant to remantadine and susceptible to oseltamivir, zanamivir, and arbidol. The influenza B virus strains were susceptible to oseltamivir, zanamivir, and arbidol. The proportion of pathogens of some ARVIs was as follows: parainfluenza viruses, 9.8%; adenoviruses, 5.5%; respiratory syncytial virus, 4.8%; and Mycoplasma pneumonia, 0.6%. There is evidence that there is a need for further monitoring of influenza viruses in Russia.     

Method for the cross protection of mice in studying the antigenic variability of the influenza virus

A modification of the method of cross protection of mice was developed for the study of influenza virus antigenic drift. This modification does not require a pre-adaptation of the virus to mouse lungs. The experiments of cross protection of immune animals carried out by the modified method demonstrated antigenic variability of the influenza A virus strains (H3N2) isolated in 1968-1983. Immunologically significant differences between influenza A/Hong Kong/68/ and A/Victoria/36/72 virus strains were detected. Subsequently, with isolation of more influenza virus strains immunologically significant differences were found between A/Victoria/36/72 and A/Leningrad/42/75 (an analogue of A/Scotland/840/74) strains, A/Leningrad/42/75 and A/Leningrad/399/76 (an analogue of A/Victoria/3/75) strains. The differences between influenza A/Texas/1/77 and A/Leningrad/527/80 (an analogue of A/Bangkok/1/79), A/Leningrad/385/80 (an analogue of A/Bangkok/1/79), and A/Leningrad/50/83, (an analogue of A/Philippines/2/82) strains were not immunologically significant.     

Serological diagnosis of influenza B virus infection: comparison of an enzyme-linked immunosorbent assay and the hemagglutination inhibition test.

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to influenza B virus was compared with that of the hemagglutination inhibition test on acute- and convalescence-phase sera obtained from adults and children infected with influenza B virus. Two whole virus, tissue culture-grown antigen preparations were used in the ELISA, influenza B/West Virginia/81 and influenza B/Hong Kong/72. Four antigens were used in the hemagglutination inhibition test. These included the tissue culture-grown whole virus antigens that were used in the ELISA. In addition the standard egg-grown antigens, influenza B/Singapore/79 and influenza B/Hong Kong/72, were included for comparison. The ELISA antibody titer was significantly correlated to the hemagglutination inhibition antibody titer, and 10 of 10 adults and 17 of 21 children infected with influenza B had fourfold antibody increases as detected by ELISA with either antigenic type of tissue culture-grown whole virus. Increases in geometric mean antibody titers of 16- to 71-fold were detected by ELISA. Increases in geometric mean antibody titers of 3- to 10-fold were detected by hemagglutination inhibition depending on the type of antigen utilized. We found that ELISA with whole virus antigens was a sensitive and specific test for the detection of antibody to influenza B virus.     

Selection of antigenic variants of H1N1 influenza viruses by means of monoclonal antibodies

Six different monoclonal antibodies to influenza A/Brazil/11/78 virus hemagglutinin were used for selection of antigenic variants of H1N1 viruses: A/USSR/090/77 and A/black-headed gull/ Kaz . SSR/470/79. The group-specific monoclonal antibody completely neutralized the infective activity of the parental viruses (dilutions 1:5 to 1:640). Two antigenic variants of wild type viruses were obtained using cross-reactive antibody. A comparative study of the antigenic structure, biological properties, and peptide maps of the heavy chain of the original viruses, antigenic variants, and some epidemic H1N1 strains was carried out. The selected variants of A/black-headed gull/ Kaz . SSR/470/77 and A/USSR/090/79 viruses were shown to be similar to epidemic H1N1 strains isolated in 1953 and 1978.     

Epidemic strains influenza viruses A and B in the 2005-2006 season in Russia

Investigations indicated that the epidemic upsurge of influenza morbidity in the 2005-2006 season in Russia was caused by the active circulation of influenza viruses A and B. The Center for Ecology and Epidemiology of Influenza, D. I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, studied 182 epidemic strains. A hundred and thirteen influenza viruses A(H3N2) were similar to the reference A/California/07/2004 or were its antigenic variants. Thirteen influenza virus A(H1N1) strains that were antigenic variants of the reference A/New Caledonia/20/99 were isolated in sporadic cases. Influenza viruses B were similar to B/Malaysia/2506/2004--lineage B/Victoria/2/87). All the strains were isolated in the MDCK cell culture. Comparative study of the sensitivity of the chicken embryo (CE) and MDCK isolation system to the 1999-2006 epidemic strains showed that CE tropism was least pronounced in influenza viruses A(H3N2). Analysis of the 2002-2006 strains demonstrated that influenza viruses A reacted actively with human erythrocytes of the blood groups 0(I) and A(II) and very slightly with chicken ones. Eighty-five influenza virus A(H3N2) strains from the 2005-2006 epidemic season were investigated for rimantadine susceptibility. The frequency of rimantadine-resistant influenza virus A(H3N2) strains was 38.0%. Studies of 79 paired sera from patients revealed a rise of antibodies to influenza viruses A(H3N2) and B in 25.9-33.3 and 20.7-23.8% of cases, respectively. There was an increase in antibodies to influenza viruses A and B in the sera collected from donors in Moscow and its region in September 2005 to June 2006.     

Hemagglutinin-neuraminidase sequence and phylogenetic analyses of mumps virus isolates from a vaccinated population in Singapore

During 1999-2000, a sustained mumps outbreak in the highly vaccinated population in Singapore was attributed to vaccine failure associated with the Rubini vaccine strain. To explain this phenomenon, the complete nucleotide and amino acid sequences of the hemagglutinin-neuraminidase (HN) gene of eight mumps virus isolates from patients with parotitis in Singapore were determined and compared with those of known vaccine strains. Phylogenetic trees constructed on the basis of HN nucleotide and amino acid sequences showed that the Singapore mumps virus isolates were more closely related to the Urabe strain and belonged to a different cluster from the Rubini and Jeryl-Lynn strains. The Rubini vaccine showed only approximately 93% nucleotide and approximately 96% amino acid sequence similarity to Urabe and Singapore isolates. Compared with the vaccine strains, six of the eight isolates lacked the extracellular glycosylation site at residues 400-402. Other significant amino acid disparities (e.g., at residue 354) may also affect the antigenic properties of the HN protein. These findings suggest that the evolution and adaptation of the currently circulating mumps virus strains in the community has led to the emergence of genetically distinct viral strains. The low vaccine efficacy of the Rubini strain represents a major reason for the recent mumps resurgence and failure of mumps immunization in Singapore.     

Antigenic determinants of the hemagglutinin of influenza virus type B

A comparative immunological analysis of 3 influenza type B virus strains, B/Lee/40, B/M/8/52, and B/Victoria/70 was made. The presence of at least 3 antigenic determinants in hemagglutinins of the viruses under study was established by the method of selective adsorption. One of the determinants was type-specific and was shared by all the three strains, the other two determined the group- and strain-specificity of the viruses studied.     

Antibody Response After Influenza Immunization in Renal Transplant Patients Receiving Cyclosporin A or Azathioprine

Nineteen renal transplant recipients receiving cyclosporin A and prednisone, eight kidney recipients receiving azathioprine and prednisone, and 12 healthy volunteers were immunized with 0.5 ml of trivalent influenza vaccine containing A/Bangkok/1/79 (H₃N₂), A/Brazil/11/78 (H₁N₁), and B/Singapore/222/79. Nine patients (47%) in the cyclosporin A group and five (63%) in the azathioprine group showed fourfold rises in titer to at least one virus strain compared with 12 (100%) in the control group.     

Genomic and antigenic analysis of strains of influenza A virus (H1N1) isolated in 1982-1983: approaches to the choice of the optimal vaccine strain

Features of the genome and antigenic specificity of hemagglutinin of some influenza A (H1N1) virus strains circulating in the epidemic period of 1982-1983 were studied comparatively. Analysis of the genome of the isolates under study in comparison with that of the reference A/England/333/80 strain and with each other has established changes not only in the genes coding for hemagglutinin and neuraminidase but also most of the genes coding for unglycolysed proteins. The antigenic specificity of hemagglutinin of the isolates under study examined with rat antisera and monoclonal antibodies was found to be quite dissimilar. Hemagglutinin of the A/Dunedin/27/83 strain induced antibodies capable of reacting predominantly with the homologous strain whereas antibodies to hemagglutinin of the A/Leningrad/16/16/82 and A/Chile/1/83 strains had a wide spectrum of antigenic specificity and neutralized well the hemagglutinin of different variants of influenza virus of H1N1 serotype circulating at that time. Among the 1982-1983 isolates studied, the A/Leningrad/16/16/82 strain was selected which, by its hemagglutinin properties, is optimal for preparation of inactivated vaccine, as was confirmed by the study of an experimental batch of such vaccine in volunteers.     

Comparative study on the properties of influenza B viruses isolated in 1959-1974]

The biological properties and antigenic structure of the following influenza B virus strains were compared: Moscow/Likh/59, Moscow/106/62, Moscow/1/66, Hong Kong/5/72, Yamagata/73 and USSR/01/4. The influenza B virus strains isolated in 1972-1974 differed by hemagglutinin from those isolated in 1959-1966. The variability of hemagglutinin correlated with that of neuraminidase. Yamagata/73 virus occupied a special position both with regard to the structure of surface antigens and to some other biological properties. Therefore, the possibility of occurrence in the USSR of virus strains similar to Yamagata/73 cannot be ruled out.