Internal epidemic influenza virus proteins: isolation and investigation

The internal influenza virus proteins M1 and RNP free from surface protein impurities were isolated from subviral particles (virions free from HA and NA ectomenes). The spikeless particles had no propensity to aggregate in the solution at pH 5.0 as compared with native viruses. The subviral particles of B/Hong Kong/330/01 influenza virus, which belonged to B/Victoria/2/87-lineage, were obtained by proteolytic treatment with the enzyme bromelain under the same conditions as in cases of influenza B viruses of B/Jamagata/16/88 lineage. A chromatographic analysis of the tryptic hydrolyzates obtained for matrix (M1) proteins of A(H1N1) and A(H3N2) influenza viruses revealed differences that were greatest between the protein M1 molecules isolated from influenza viruses of different subtypes of hemagglutinine. These findings suggest there are variations in the structure of this conservative internal viral protein M1 during evolution.     

Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983

During 1988-1989 two highly distinct antigenic variants of influenza type B were recognized in hemagglutination-inhibition tests with postinfection ferret serum. These viruses were antigenically related to either B/Victoria/2/87, the most recent reference strain, or B/Yamagata/16/88, a variant that was isolated in Japan in May 1988. All influenza B viruses isolated in the United States during an epidemic in the winter of 1988-1989 were antigenically related to B/Victoria/2/87. However, in several countries in Asia, both B/Victoria/2/87-like viruses and B/Yamagata/16/88-like viruses were isolated. Sequence analysis of the hemagglutinin (HA) genes of several influenza B isolates from 1987 to 1988 indicated that the HA1 domains of the B/Yamagata/16/88-like viruses and B/VI/87-like viruses isolated in 1988 differed by 27 amino acids. Evolutionary relationships based on this sequence data indicated that the B/Yamagata/16/88-like viruses were more closely related to epidemic viruses from 1983 (B/USSR/100/83-like viruses) than to more recent reference strains such as B/Victoria/2/87. All other Asian strains, as well as selected isolates from the United States in 1988, were confirmed by sequence analysis as being genetically related to B/Victoria/2/87. These data provide clear evidence that two parallel evolutionary pathways of influenza type B have existed since at least 1983 and that viruses from each of the separate lineages were isolated from cases of influenza B in 1988. This finding is similar to earlier observations for type A H1N1 and H3N2 influenza viruses.     

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 and characteristics of monoclonal antibodies to influenza virus types A and B

Monoclonal antibodies (MCA) to influenza type A (10F) and B (5H and 6H) viruses have been prepared. By immunoblotting method, MCA 10F were found to be specific for NP-protein of influenza A virus, and MCA 5H and 6H to be specific for hemagglutinin of influenza B virus. It was established that the 10F clone interacted with all the investigated influenza A virus strains with different antigenic formulae (H1N1, H2N2, H3N2) and could be used for typing of this virus type. Clones 5H and 6H react specifically with hemagglutinins of influenza B viruses isolated in 1940, 1979, and 1983 which makes them useful for diagnosis of influenza B.     

Evolutionary pattern of influenza B viruses based on the HA and NS genes during 1940 to 1999: origin of the NS genes after 1997

Summary.  Phylogenetic analysis was carried out for genes encoding hemagglutinin (HA) (24 new and 25 previously reported sequences) and nonstructural proteins (NS) (22 new and 14 previously reported sequences) of influenza B virus isolates obtained from 1940 to 1999. Two antigenically and genetically distinct HA lineages are presently known to exist. Divergence into these two lineages was estimated to have occurred around 1969. Phylogenetic analysis of NS genes revealed that their phylogenetic relationships were not linked to the two HA lineages but suggested that reassortment of viral genes between the viruses of two HA lineages had occurred. In addition two distinct NS lineages which were not linked to the two HA lineages were observed. Viruses isolated after 1997 formed their own lineage in combination with B/Houston/84 while other virus isolates obtained from 1973 to 1995 comprised the other NS lineage. Accepted May 26, 1999 Received March 26, 1999     

The influence of the isolation technique of influenza virus nucleoprotein on its antigenic properties

ELISA has been used to study the antigenic properties 1. of influenza virus nucleoprotein (NP-1) isolated from virions with the help of preparative polyacrylamide gel electrophoresis (PAGE); 2. of virion ribonucleoprotein (NP-2), and 3. of NP structures prepared by dissociation of ribonucleoprotein into RNA and protein in sucrose gradient containing NaCl (NP-3). The investigation of immunologic cross-reactivity has shown complete identity of NP-2 and NP-3 and their striking difference from NP-1. In contrast to NP-2, NP-3 was not contaminated by other virus antigens, it was a good immunogen and could be used for preparation of monospecific antisera of high titre. NP-1 did not induce a high antibody response,however, like NP-2 and NP-3, it retained its capacity to react with antisera to native virus. Owing to its simple production and high yield, this protein can be used in serodiagnosis for testing the antibody level against NP-protein in convalescent sera.     

Analysis of the mechanism of influenza B virus inactivation by guinea pig serum

Summary Normal guinea pig serum lacking detectable antiviral antibody inactivated influenza B virus via the classical complement pathway [12]. This virus inactivation appeared to result from the steric hindrance of HA activity by the association with the virus of serum proteins presumed to be complement components. Trypsin digestion of the associated proteins fully restored the HA activity but not infectivity. It was found that the virus underwent minor disruption of the envelope and degradation of M₁ protein and genomic RNA.     

Further isolation and characterization of temperature-sensitive mutants of influenza virus.

We have assigned 34 temperature-sensitive (ts) mutants of the WSN strain of influenza virus into seven nonoverlapping recombination-complementation groups by conducting pairwise crosses. A single gene of the viral genome was affected in 27 mutants while six were double mutants and one was a triple mutant. The synthesis of virion RNA in mutant-infected cells at the nonpermissive temperature was studied by the incorporation of [³H]uridine in the presence of actinomycin D into an acid-insoluble product. This method separated mutants into two classes. Mutants belonging to Groups I, II, III, and V were RNA^?, while those belonging to Groups IV, VI, and VII were RNA⁺. The results of temperature shift-up experiments suggested that all four groups of RNA^? mutants were defective in replicating virion RNA. Production of serologically or functionally active subviral components (ribonucleoprotein, hemagglutinin, and neuraminidase) by RNA^? mutants at the nonpermissive temperature was variable. RNA⁺ mutants produced a normal amount of these components, except Group IV mutants in which the production of functional hemagglutinin and neuraminidase was greatly depressed.     

Polymorphism insertion/deletion of apolipoprotein B gene: effect on lipid levels in obese patients

Apolipoprotein B (apoB) is the major proteic component of LDL, VLDL and chylomicrons. Numerous polymorphisms of the apolipoprotein B gene have been described. Particularly, a polymorphism of insertion/deletion located in the coding part of the signal peptide of apoB, associated with modifications of lipid concentrations and the risk of cardiovascular disease, has been reported in the general population. Since obesity is frequently associated with dyslipidemias, the aim of our study was to assess the effect of the insertion/deletion polymorphism of the apolipoprotein B gene on lipid levels in obese subjects. 234 unrelated caucasian obese subjects (74 men and 160 women, aged 39.3 +/- 10.5, BMI : 32.8 +/- 4.7) were recruited. The insertion/deletion polymorphism was determined by electrophoresis in polyacrylamide gels after PCR amplification. The relative frequencies of the Ins and Del alleles were 0.71 and 0.29 respectively. These frequencies were similar to those found in other Caucasian populations. In the whole population, individuals with the Del/Del genotype had significantly higher total-cholesterol to HDL-cholesterol ratios (p = 0.004), LDL-cholesterol to HDL-cholesterol ratios (p = 0.01) and TG-VLDL levels (p < 0.05). They also showed a tendency for higher triglyceride levels (p = 0.09) and lower HDL-cholesterol, apolipoprotein AI and LpAI levels. The allele deletion results in the absence of three amino acids (Leu-Ala-Leu) in the signal peptide of apo B. In the obese people, these structural changes may have some effect on lipid metabolism and cause variation in serum lipid concentrations.     

Analysis of the circulation of hepatitis A virus in Argentina since vaccine introduction

Hepatitis A virus (HAV) has shown intermediate endemicity in Argentina, but its incidence has decreased since vaccine introduction in 2005. Environmental surveillance was conducted in five rivers from Argentina from 2005 to 2012, complementing clinical information. HAV detection decreased since 2005, although its circulation continues, maintaining viral diversity but not undergoing antigenic drift. Most sequences belonged to subgenotype IA, closely related to Argentinean clinical sequences, but one belonged to proposed subgenotype IC, previously undetected in the country. Environmental surveillance might contribute to monitoring the single-dose vaccination schedule, representing not only strains causing disease but also the circulating population and the viral introductions.     

Evolutionary analysis of human-origin influenza A virus (H3N2) genes associated with the codon usage patterns since 1993

This study investigated genetic variations in eight major genes (hemagglutinin, HA; neuraminidase, NA; matrix protein, MP; non-structural protein, NS; nucleoprotein, NP; polymerase, PA; PA basic protein 1, PB1; and PA basic protein 2, PB2) of the influenza A virus subtype H3N2 (A/H3N2) to determine the evolutionary pattern in codon bias. A total of 6,881 sequences isolated between 1993 and 2010 were used. The relative synonymous codon usage (RSCU) and G+C% content at the three codon positions were analyzed by calculating the codon substitution patterns were analyzed by calculating the percentage of synonymously substituted codons (SSCs) and that of codons substituted to the same codon within each synonymous codon group (EMC) between 1993 and subsequent years. In the multivariate analysis of RSCU, we observed directional changes in HA, NA, PB1, and PB2, and these changes were significantly correlated with the variation in the G+C contents at the first (GC_1st) and second (GC_2nd) codon positions over time. These directional changes in HA and NA appear to affect their antigenic characteristics by altering their SSCs gradually, and NP, PA, PB1, and PB2 genes also continuously changed their substitution patterns by accumulating the decrements of EMC values over a long term. Our findings suggest that, in human populations, A/H3N2 viruses have gradually changed their SSCs in two external genes, HA and NA, and that these accumulated alteration patterns may result in the antigenic changes over time. Moreover, A/H3N2 viruses also appear to change synonymous codon usage patterns in NP, PA, PB1, and PB2 genes by accumulating decrements in EMCs within synonymous codon groups over time.     

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.     

Characteristics of the biological and antigenic properties of reference strains of the influenza B virus at various periods of isolation

The results of a comparative analysis of biological and antigenic properties of reference influenza B virus strains, B/Lee/40, B/Singapore/222/79, and B/USSR/100/83, are presented. The most marked changes were found in the antigenic properties of hemagglutinin and neuraminidase. Hemagglutinins of the strains under study were found to have both common antigens and qualitatively different strain-specific determinants. The degree of intensity of immunity to the challenge B/Singapore/222/79 Pm+ virus corresponded to the intensity of antigenic relationship in the strains under study.