Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion.

The spike glycoprotein of influenza C/Johannesburg/1/66 was isolated in a soluble form by digestion of MDCK cell-grown virions with bromelain. The whole ectodomain of the glycoprotein could be recovered with an apparent molecular weight of 75,000 daltons determined in SDS-PAGE. Comparison to Triton X-100-isolated glycoprotein revealed that a C-terminal peptide of 3000-4500 daltons must have remained in the viral membrane. When purified by sucrose density gradient centrifugation the glycoprotein sedimented with a sedimentation coefficient of 10 S, indicating a molecular weight of 206,000 daltons, which is consistent with a trimeric structure of the spike molecule. The trimeric form was stabilized in sucrose gradients by Ca2+ ions. Bromelain digestion of virions with uncleaved glycoprotein, grown in MDCK cells without trypsin, produced two disulphide-linked subunits with similar electrophoretic mobilities in SDS-PAGE to the biologically active glycoprotein. The smaller subunit differed from the product cleaved in vivo (gp 30) by the presence of an additional arginine residue at the N-terminus. The soluble glycoprotein appears to possess both receptor-binding and receptor-destroying enzyme activities, as isolated glycoprotein inhibited hemagglutination of intact influenza C virions and showed RDE activity in an in vitro test. Glycoprotein exposed to low pH, which was sensitive to trypsin digestion, also demonstrated both these biological activities. Glycoprotein-mediated hemolysis could not be observed.

Nucleotide sequence analysis of the HA1 coding portion of the haemagglutinin gene of swine H1N1 influenza viruses.

The nucleotide and deduced amino acid sequences coding for the HA1 portion of the haemagglutinin (HA) genes of three swine influenza viruses were determined and compared with published HA sequence data for human H1N1 influenza viruses. Sequence differences between the classic swine influenza HAs sw37 (A/swine/29/37) and NJ76 (A/New Jersey/11/76) were randomly distributed in the molecule without being confined to antigenic sites. In contrast, sequence differences between the HAs of sw37 and the antigenically atypical strains sw38 (A/swine/Northern Ireland/38) and sw39 (A/swine/Cambridge/39) were clustered in hypervariable regions, similar to the pattern of changes that was present between sw37 and the human strains PR834 (A/PR/8/34) and WSN33 (A/WSN/33). Sequence homologies of the European swine influenza strains (sw38, sw39) were higher with the HAs of the human strains (PR834, WSN33) than with the classic swine influenza HAs (sw37, NJ76). Phylogenetic analysis showed that the HA genes of these two European swine influenza strains emerged from a different evolutionary lineage of H1 HAs than the HAs of classic swine influenza strains.

The persistent variant of influenza C virus carries one characteristic point mutation in RNA segment 1.

Influenza C/ Ann Arbor/1/50-pi(C/AA-pi) virus causes persistent infections in MDCK and Wi38 cells, but sets limited, wild-type like infections in other cells. Concluding that persistence itself it dependent on the host environment, we determined the nucleotide sequence of the C/AA-pi analogous gene to the basic polymerase 2 (PB2) of influenza A virus, which is known to be a determinant for the host range. C/AA-pi and the parental wild-type virus (C/AA-wt) have 16 nucleotides in common that are different to a previously published PB2 sequence (C/JJ/50). These variations, which are probably due to divergent passages histories, are scattered along the sequence and are partially found in another published isolate (C/Berlin/1/85). One single mutation, however, is unique to the persistent variant. Nucleotide 28 mutated from T to C which leads to a change of amino acid 3 from Leu to Phe. This substitution is stably associated with the persistent phenotype throughout multiple passages in different cells lines and eggs and cannot be found in any other influenza C viruses.

A persistent variant of influenza C virus fails to interact with actin filaments during viral assembly.

C/AA-pi virus, a variant of influenza C/Ann Arbor/1/50 virus, establishes persistent infections in MDCK cells, characterized by low levels of progeny production. During viral assembly, nucleoprotein (NP) was found homogeneously distributed over cytoplasmic and nuclear compartments and matrix (M) protein was likewise localized in a barely structured fashion. In contrast, infections with nonpersistent influenza A, B and C viruses produced cytoplasmic granular structures, which typically consisted of colocalized NP and M proteins. Studies on the in vitro interaction between NP and M proteins revealed identical binding capacities comparing influenza C wild-type virus with the persistent variant. Cytochalasin D treatment of infected cells demonstrated that NP protein of the wild-type virus, but not of the persistent variant, was distinctly associated with cellular actin filaments. Moreover, the assembly characteristics of wild-type virus were modulated in the presence of recombinant persistent-type NP protein towards a behaviour similar to persistent infection. Cell type specificity was particularly illustrated in C/AA-pi virus-infected Vero cells, which did not support viral persistence, but produced granular wild-type-like complexes. Thus, interaction between NP, M and actin proteins (i) is a basic part of the viral assembly process, (ii) is dominantly modulated by NP protein and (iii) is specifically altered in the case of persistent infection.

Persistent infection with an influenza C virus variant is dominantly established in the presence of the parental wild-type virus.

Two influenza C viruses were used for double-infection experiments to investigate the dominance of their phenotypes. The wild-type virus (C/AA-wt) had been characterized by its short-lived productive cycle, whereas a distinct variant derived from it (C/AA-pi) was demonstrated to persist in long-term passages of infected MDCK cultures. Here we show that the persistent virus C/AA-pi is capable of replicating in the presence of abundant amounts of wild-type virus: the persistent virus could be diluted to 10(-9) within wild-type inoculum, still developing a stable form of persistence. This behaviour was reflected by permanent virus release and by continuous enzymatic activity of the viral HEF glycoprotein in infected cells. All long-term cultures tested remained positive for viral NS protein and vRNA. On the vRNA level, it was shown that viral segments originated from the persistent-type genome, while wild-type vRNAs were not maintained after double-infection. Thus, the genotype of the persistent variant was dominantly selected in serial passages. These results indicate a specific intracellular advantage of persistent influenza C virus over the parental wild-type.

Inhibition of influenza C viruses by human MxA protein.

Human MxA protein was analyzed for its ability to inhibit the replication of different influenza C viruses. Three laboratory derivatives of viral strain C/Ann Arbor/1/50 were investigated, namely the parental wild-type virus C/AA-wt, the persistent variant C/AA-pi and the highly cytopathogenic variant C/AA-cyt. In addition, strain C/Paris/214/91 isolated from an influenza patient was used. Multiplication of all four viruses was suppressed in MxA-expressing Vero cells, as indicated by a decrease in viral RNA synthesis, viral protein synthesis, virion production and induction of a cytopathic effect. Inhibition correlated with the level of MxA expression. Furthermore, inhibition was independent of cell clone-specific differences in expression of virus receptors, as demonstrated by receptor reconstitution experiments. Thus, human MxA protein has antiviral activity against influenza C viruses.

Sleep loss reduces diurnal rhythm amplitude of leptin in healthy men.

The aim of the current study was to investigate the effects of sleep loss on the diurnal rhythm of circulating leptin levels. An indwelling forearm catheter was used to sample blood at 90-min intervals for a total of 120 h, which included 88 h of sustained sleeplessness, in 10 healthy men. The diurnal amplitude of leptin was reduced during total sleep deprivation and returned toward normal during the period of recovery sleep. This finding provides evidence that sleep influences the nocturnal leptin profile, and may have implications for the understanding of the role of sleep in metabolic regulation and the aetiologies of obesity and the night eating syndrome.

Nucleotide-specific PCR for molecular virus typing.

Nucleotide sequence studies detected a double-point mutation in the genomic RNA segment 4 (nt 871 and 872) of the persistent variant C/AA-pi of influenza C/Ann Arbor/1/50 virus. The 3'-end-points of two distinct PCR primers were positioned exactly at this genome location and thereby adjusted the priming determinant complementary to the varied strain or to its wild-type counterpart. Consequently, positive RT-PCR products strictly referred to one of the two viruses examined, in both cases, using either virion or infected-cell RNA templates. Artificial virus mixtures could easily be distinguished by this method in a subsequent qualitative gel analysis. PCR annealing conditions and control reactions were optimized, for the monitoring of influenza virus isolates throughout multifold passages. Thus, sequence diversity in just two neighbouring nucleotides is sufficient to determine whether or not successful PCR amplification takes place, and this method can be used as a reliable means of virus strain differentiation.

Flow cytometric analysis of virus-infected cells and its potential use for screening antiviral agents.

Virus-infected cells were analyzed using multiparameter flow cytometry. Two virus-cell systems were investigated: HSV-1-infected VF cells and influenza C virus JHB/1/66-infected MDCK cells. Analysis included the measurement of the appearance of virus specific antigens. On individual cells, with polyclonal antibodies, antigens were first detected at 12 h p.i., and the numbers of labeled cells were followed up to 96 h p.i. The efficacy of four antiviral agents was tested with this system. The results were in good agreement with those of plaque reduction tests and indicated that this new method may be extremely useful for the correlation of viral and cellular events with antiviral activity. Finally, it was demonstrated that infected cells in both systems have a considerably greater volume than non-infected cells.

Influenza-C-virion-associated RNA-dependent RNA-polymerase activity.

The addition of detergent-solubilized influenza C virus to a reaction mixture containing ATP, CTP, GTP, [3H]UTP, and Mg2+ leads to the synthesis of an acid-insoluble, radioactive product, which is ribonuclease-sensitive. The dinucleoside monophosphate ApG clearly enhances the reaction rate, a fact which indicates that influenza C viruses follow the same strategy of transcription as influenza A and B viruses, the other members of the orthomyxovirus family.

Experimental infection with a persistent influenza C virus variant leads to prolonged genome detection in the chicken lung.

A persistent variant of influenza C virus was used to infect chickens by intraamniotic (i.a.) inoculation. The infected hatchings were analyzed for virus production in different tissues and for the continuous presence of viral RNA genomes. The permissiveness for infection was demonstrated primarily for the chicken lung, besides other organs. Viral antigens could be detected by indirect immunofluorescence staining for a period of 8 days and reisolates were obtained mainly at early time points post infection (p.i.). Nested reverse transcription-polymerase chain reaction (RT-PCR) directed to 3 genomic sequences was positive at least until day 53, whereby no distinct end point was determined. These experiments provide first evidence for the long-term stability of influenza C virus RNA segments in vivo.

Safety and efficacy of high-dose melphalan and auto-SCT in patients with AL amyloidosis and cardiac involvement.

In Ig light chain (AL) amyloidosis, cardiac involvement is associated with worse prognosis and increased treatment-related complications. In this retrospective cohort study, we assessed survival, hematologic and cardiac responses to high-dose melphalan and auto-SCT (HDM/SCT) in patients with AL amyloidosis and cardiac involvement, stratified by cardiac biomarkers brain natriuretic peptide and Troponin I, analogous to the Mayo cardiac staging. Forty-seven patients underwent HDM/SCT based upon functional measures; six patients had modified cardiac stage I disease, seventeen had modified cardiac stage II disease and twenty-four had modified cardiac stage III disease. Treatment-related mortality was 4% for all patients and 8% for patients with stage III disease. Three-year survival was 88% and EFS was 47%; these did not differ by stage. By intention-to-treat analysis, 27% of patients achieved a hematologic complete response and 32% a very good partial response, of whom 70 and 45%, respectively, have not required additional therapy at 36 months. Cardiac response was achieved in 53% of patients. We conclude that with appropriate patient selection and a risk-adapted treatment approach, HDM/SCT is safe and effective in patients with AL amyloidosis and cardiac involvement.

In-vitro activity of fleroxacin against Chlamydia trachomatis.

The in-vitro activities of fleroxacin, tetracycline, erythromycin and clindamycin against two clinical isolates of Chlamydia trachomatis were compared. The MIC of fleroxacin (4.0 mg/l) was comparable to that of clindamycin (2.0 mg/l), but was higher than the MICs of both tetracycline (0.4 mg/l) and erythromycin (0.2-0.4 mg/l). Repeated passaging of C. trachomatis in the presence of sub MIC concentrations of fleroxacin did not affect the MIC and resulted in a rapid and complete loss of inclusions. The effect of combining fleroxacin with the other antibiotics was investigated by chequerboard titrations of pairs of antibiotics. The mean fractional inhibitory concentration index was calculated for each combination. Results showed indifference for the combinations fleroxacin/tetracycline, fleroxacin/erythromycin and fleroxacin/clindamycin and synergism for the combination tetracycline/clindamycin.

[Echo-6-virus as the pathogen of hand, foot and mouth exanthema]. / Echo-6-Viren als Erreger des Hand-Fuss-Mund-Exanthems

Seven cases of typical hand-foot-mouth-disease are reported. As causative agent Echovirus Type 6 was isolated.

Time course of synthesis and assembly of influenza virus proteins.

The synthesis of viral polypeptides was analyzed in BHK-21-F cells infected with the WSN strain of influenza virus at various times in the growth cycle. The relative amounts of polypeptides P, HA, NP, and NS did not change markedly between early and late times in the growth cycle; however, there was a progressive increase in the relative amount of the M polypeptide at later time points. In cell fractionation experiments, the patterns of newly synthesized polypeptides associated with various cytoplasmic fractions remained similar throughout the growth cycle except for an increase in polypeptide M in all fractions late in the growth cycle. The HA polypeptide was chased out of cytoplasmic membranes completely 6 h after synthesis, whereas the M polypeptide was not chased effectively from such membranes. Marked differences were found in the incorporation into mature virions of polypeptides synthesized at different times in the growth cycle. Polypeptides P and NP synthesized at early times were incorporated preferentially, whereas M was synthesized and incorporated predominantly late in the growth cycle. The fact that the rates of incorporation of polypeptides into virions differed significantly from their rates of synthesis indicates that different polypeptides were assembled into virions by distinct pathways.

Influenza C virus RNA is uniquely stabilized in a steady state during primary and secondary persistent infections.

The ability to establish persistent infections in vitro and in vivo has been illustrated for different human RNA viruses. However, little insight has been gained regarding the intracellular state of viral RNA species and the regulatory processes governing their long-term continuance. In this report, primary persistence of a variant of influenza C/Ann Arbor/1/50 virus in infected MDCK cells and secondary infections in human cell lines were investigated. Different PCR and staining techniques were applied for the description of low viral loads. The RNA pattern in primary persistence indicates that viral RNA synthesis is quantitatively linked to productive and non-productive phases, with negative-strand RNA being present continuously. In single cells cultures, derived from the primary line, all clones tested were positive by nested PCR and Southern blot screening. This suggests that a true steady-state persistence of influenza C virus is established in each individual cell of the infected population. Secondary infection experiments, in terms of transfer of the persistent virus variant to different cell types, showed that a re-establishment of persistence can be accomplished in vitro. The stable persistent status remained reserved for distinct host cell lines. Hereby, vRNA is stably maintained in cell-type specific manner, whereas gene expression (e.g. HEF glycoprotein production) occurs in a variable fashion. These data point out novel characteristics in the understanding of influenza virus persistence.

Genetic relatedness between influenza A (H1N1) viruses isolated from humans and pigs.

Complete nucleotide sequences were obtained from the nucleoprotein genes of three influenza A viruses and partial nucleotide sequences were obtained from the polymerase, neuraminidase, matrix, and non-structural protein genes of four influenza A viruses that had been isolated between 1931 and 1939 from clinically sick pigs in the United States or Europe. A phylogenetic analysis of the open reading frames of nine nucleoprotein genes showed that the U.S. swine influenza virus isolates from 1931 and 1937 originated from the classic swine viral nucleoprotein lineage, whereas the European swine strains fro 1938 and 1939 were placed among the early human influenza virus nucleoprotein lineage. All the partial gene sequences obtained from the two European swine strains fro 1938 and 1939 were also more closely related to early human H1N1 reference strains than to the U.S. swine isolates from 1931 and 1937, indicating that none of the four viruses isolated from swine had acquired genes from a heterologous lineage through reassortment.

Comparative analysis of six European influenza vaccines.

Three split-virion vaccines (Vaxigrip, Begrivac, and Influsplit/Fluarix) and three subunit vaccines containing only viral surface glycoproteins (Influvac, Agrippal, and Fluvirin) available for the 1994-95 season were analysed by biological, molecular, and biochemical methods. Although all vaccines are required by health authorities to contain 15 micrograms haemagglutinin per dose of each virus strain, there were significant differences in haemagglutination titres among the examined vaccines of both types. The enzymatic activity of neuraminidase was present in all vaccines except Fluvirin. Total protein content was lower for subunit vaccines. Viral nucleoprotein was detected in all split vaccines but to varying levels according to SDS-PAGE and Western blot analyses. The ovalbumin content was low in general but was about tenfold higher for Influvac than for the other vaccines analysed. This protein may induce hypersensitive reactions among persons with severe egg allergy. All three split-virion vaccines were found to contain the matrix protein; however, it was not detected in the subunit vaccines. Differences in influenza antigen variety in currently available vaccines may affect efficacy, whereas differences in concentrations of nonviral compounds such as ovalbumin and endotoxin may lead to different postvaccination reactogenicity profiles.