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.

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.

The ORF, regulated synthesis, and persistence-specific variation of influenza C viral NS1 protein.

The open reading frame (ORF) and the regulated synthesis of the influenza C viral NS1 protein were analyzed in view of viruses possessing different biological activities. We provide evidence for a 246-amino-acid NS1-ORF, encoded by five viral strains and variants. Prokaryotic expression of the prototype NS1-ORF resulted in a product of 27 kDa, confirming the predicted molecular weight. Using an antiserum raised against recombinant NS1 protein, nonstructural proteins of wild-type virus were detected in infected cells for a limited course of time, whereas a persistent virus variant was characterized by a long-term nonstructural gene expression. As examined by infection experiments, the intracellular distribution of nonstructural protein was nuclear and cytoplasmic, whereas in NS1 gene-transfected cells, the cytoplasmic localization occurred in a fine-grained structure, suggesting an analogy to influenza A viral NS1 protein. Concerning persistent infection, NS1 protein species differing in sizes and posttranslational modifications were observed for a persistent virus variant, as particularly illustrated by a high degree of NS1 phosphorylation. Virus reassortant analyses proved the importance of the NS-coding genomic segment: the minimal viral properties required for the establishment of persistence were transferred with this segment to a monoreassortant virus. Thus the influenza C viral NS1 protein is a 246-amino-acid nuclear-cytoplasmic phosphoprotein that can be subject to specific variations being functionally linked to a persistent virus phenotype.

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.

A highly cytopathogenic influenza C virus variant induces apoptosis in cell culture.

An influenza C virus variant, C/AA-cyt, was identified as the agent responsible for highly effective induction of cytopathogenicity in MDCK cells. The cytopathogenic effect was manifested by cell rounding, cell shrinkage and foci of cell destruction leading finally to disruption of the monolayer in a virus dose-dependent manner. Virus-induced cytopathogenicity was suppressed by temperatures nonpermissive for virus replication. Maintenance of plasma membrane integrity post-infection, in connection with induction of a DNA fragmentation ladder, revealed the characteristic picture of apoptosis. In support of this, quantitative analysis demonstrated high levels of apoptosis-like oligonucleosomal DNA. The results indicate that influenza C viruses can induce programmed cell death, as formerly reported for influenza type A and B viruses.

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.