Tatiana Betakova

The NB protein is an integral component of the membrane of influenza B virus

The results of biochemical and immunoelectron microscopic studies provide evidence that the NB protein is an integral component of the influenza B virion. Its glycosylation and orientation in the membrane were shown to be equivalent to that of NB in the plasma membrane of virus-infected cells. Sensitivity to proteinase K showed that the N terminus is exterior to the virion and gold immunolabelling of freeze-fractured replicas showed that the C terminus is located in the interior of the virion. The similarities between NB of influenza B and M2 of influenza A viruses in structural features, their presence in the virion and possession of an ion channel activity suggest that, by analogy with the M2 protein, NB may also have a role in virus entry.

Associations Between Coinfection Prevalence of Borrelia lusitaniae, Anaplasma sp., and Rickettsia sp. in Hard Ticks Feeding on Reptile Hosts

An increasing number of studies reveal that ticks and their hosts are infected with multiple pathogens, suggesting that coinfection might be frequent for both vectors and wild reservoir hosts. Whereas the examination of associations between coinfecting pathogen agents in natural host–vector–pathogen systems is a prerequisite for a better understanding of disease maintenance and transmission, the associations between pathogens within vectors or hosts are seldom explicitly examined. We examined the prevalence of pathogen agents and the patterns of associations between them under natural conditions, using a previously unexamined host–vector–pathogen system—green lizards Lacerta viridis, hard ticks Ixodes ricinus, and Borrelia, Anaplasma, and Rickettsia pathogens. We found that immature ticks infesting a temperate lizard species in Central Europe were infected with multiple pathogens. Considering I. ricinus nymphs and larvae, the prevalence of Anaplasma, Borrelia, and Rickettsia was 13.1% and 8.7%, 12.8% and 1.3%, and 4.5% and 2.7%, respectively. The patterns of pathogen prevalence and observed coinfection rates suggest that the risk of tick infection with one pathogen is not independent of other pathogens. Our results indicate that Anaplasma can play a role in suppressing the transmission of Borrelia to tick vectors. Overall, however, positive effects of Borrelia on Anaplasma seem to prevail as judged by higher-than-expected Borrelia–Anaplasma coinfection rates.

Monoclonal anti-idiotypic antibodies mimicking the immunodominant epitope of influenza virus haemagglutinin elicit biologically significant immune responses.

The MAb IIB4 recognizes an immunodominant epitope on influenza virus haemagglutinin (HA) which is shared by different strains of the human subtype H3. This epitope includes amino acids 198, 199 and 201, as determined by selection of IIB4 escape mutants, and is involved in haemagglutination-inhibition (HI) and virus-neutralization (VN). We have developed anti-idiotypic MAbs (Ab2) that mimic the IIB4 epitope. Two Ab2, 78 and 464, completely inhibited binding of MAb IIB4 to the virus. Nucleotide sequences of VL- and VH-encoding regions were determined for IIB4 and both Ab2. VH of IIB4 and 78 belong to the same IgG family (VII) and show high nucleotide identity (89%). Conversely, VH and VL sequences of both internal image-bearing Ab2 revealed lower degrees of identity (61 and 50%, respectively). Ab2 were used for syngeneic immunization to elicit polyclonal Ab3 responses. Like Ab1, Ab3 immunoprecipitated viral HA and displayed HI and VN activity. The different VN activity of anti-78 and anti-464 in vitro correlated with the affinities of their corresponding Ab2 to IIB4. In vivo immunization with either Ab2 protected 15-37% of mice against lethal influenza infection or delayed dying. In contrast to VN activity of Ab3 in vitro, there was no significant difference between the protection of mice induced by Ab2 78 and 464. We demonstrate, for the influenza model, that active immunization with a single influenza virus HA epitope in the form of its internal image leads to partial protection in vivo.

Using nested RT-PCR analyses to determine the prevalence of avian influenza viruses in passerines in western Slovakia, during summer 2007

The prevalence of avian influenza virus (AIV), together with the distribution of different AIV subtypes, was studied in migratory waterfowl and terrestrial birds caught in western Slovakia during summer 2007. Both oropharyngeal and cloacal swabs were collected. Screening of samples revealed that 18% of oropharyngeal and 18% of cloacal samples were positive for AIV. Samples from both the oropharynx and cloaca were positive in only 6.6% of cases. A total of 10 different subtypes of haemagglutinin (H2, H3, H4, H6, H7, H9, H10, H11, H12, and H13) and 4 different subtypes of neuraminidase (N1, N2, N3, and N5) were detected in 32 samples from this location. The most abundant subtypes of HA in the samples were H12 and H9 (25% each), followed by H11 and H10 (15% each), and H13 (9%). There were 3 cases where different AIV infections were detected in oropharyngeal and cloacal samples originating from the same bird (H13N1 and H12N5; H13N3 and H9N5; H10N2 and H9N5 in the oropharynx and cloaca, respectively).

Prevalence of avian influenza viruses, Borrelia garinii, Mycobacterium avium, and Mycobacterium avium subsp. paratuberculosis in waterfowl and terrestrial birds in Slovakia, 2006

The prevalence of Borrelia, Mycobacteria and avian influenza virus (AIV) infections, together with the distribution of different AIV subtypes, was studied in migratory waterfowl and terrestrial birds trapped in three localities in Slovakia during 2006. Samples obtained from waterfowl captured in the Senianske Ponds area of Eastern Slovakia showed the highest diversity of AIV isolates. A total of 13 different subtypes were detected in 19 samples from this location (H1N2, H2N2, H3N2, H6N6, H7N6, H9N2, H9N5, H9N6, H10N5, H10N6, H12N6, H13N6, and H16N6). H3N5 virus was detected in 50% of passerines testing positive for AIV in the Parizske Wetlands, with H7N2, H9N2, H9N5, H12N1, and H13N2 infections also recorded at this locality. H9N5 virus predominated in passerines captured at Trnava Ponds, with isolates H1N6, H6N5, H7N2, H7N6, H10N3, and H10N6 also detected at this location. There were five cases where different AIV infections were detected in oropharyngeal and cloacal samples originating from the same bird (H13N6 and H1N2; H10N5 and H12N6; H9N5 and H6N5; H10N6 and H7N6; and H9N2 and H3N5 in the oropharynx and cloaca, respectively). Between 21% and 52% of captured birds tested positive for Borrelia burgdorferi sensu lato, with the proportion infected depending on bird species and locality. Samples were characterized by polymerase chain reaction-restriction fragment length polymorphism analysis and identified as Borrelia garinii species (either B/B′ or R/R′ pattern). Mycobacteria were detected in 42% and 26% of waders captured at Senianske Ponds and marsh-dwelling passerines captured in the Parizske Wetlands, respectively. Interestingly, forest-dwelling passerine species caught in the Trnava Ponds region were tested negative for Mycobacteria.

The strong positive correlation between effective affinity and infectivity neutralization of highly cross-reactive monoclonal antibody IIB4, which recognizes antigenic site B on influenza A virus haemagglutinin.

Monoclonal antibody (MAb) IIB4 displays a rare combination of virus neutralization (VN) activity and broad cross-reactivity with influenza A virus strains of the H3 subtype isolated in a period from 1973 to 1988. The epitope of this antibody has been identified as around HA1 residues 198, 199 and 201. Here we report that residues 155, 159, 188, 189 and 193 also influence the binding of this antibody. We have used this antibody to study the relationship between antibody affinity and VN activity. Using one MAb and a single epitope on the haemagglutinin (HA) of different influenza viruses we found a strong positive correlation between effective affinity and VN activity of MAb IIB4. A 10-fold increase in effective affinity corresponded to the 2000-fold increase in VN titre. It follows from the law of mass action that for an effective affinity K=9x10(8) l/mol, 50% VN was achieved at approx. 10% occupation of HA spikes with antibody. In contrast, for an effective affinity K=6x10(7) l/mol, to achieve 50% VN, occupation of up to 98% of HA spikes was required. An effective affinity about K=6x10(7) l/mol thus represents the limiting value for VN because a further decrease in the affinity cannot be compensated by a higher concentration of antibody.

Influenza A virus replication is inhibited in IFN-λ2 and IFN-λ3 transfected or stimulated cells.

Interferons lambda (IFN-λ) are the most recently defined members of the class III cytokine family. To investigate whether IFN-λ2 and IFN-λ3 displayed antiviral activity against influenza A virus (IAV), a number of cell lines induced with IFNs - as well as two established cell lines (A549-IFN-λ2 and A549-IFN-λ3) - were infected with IAV. Our results indicate that IFN-λ2 has statistically significant antiviral activity in A549-IFN-λ2 (P=0.0028) although less so than IFN-λ3, which reduced viral titer to 10% (P<0.0001). The reverse was observed for cells treated with IFNs, with IFN-λ2-treated A549 cells inhibiting IAV infection more efficiently than IFN-λ3-treated A549 cells. The antiviral effect on IFN-stimulated cells was most apparent on Vero cells (compared with MDCK and HeLa). Both IFNs significantly inhibited IAV replication and inhibition was observed in a dose-dependent manner, with an optimal IFN concentration of 20 ng/ml. IFN-λ2 was more potent than IFN-λ3 on Vero cells while IFN-λ3 appeared more efficient than IFN-λ2 on MDCK and HeLa cells.

Monoclonal antibodies demonstrate accessible HA2 epitopes in minor subpopulation of native influenza virus haemagglutinin molecules

SummaryHaemagglutinin (HA) was detected on the surface of influenza virus infected cell with monoclonal antibodies (MAbs) against both HA glycopolypeptides, HA1 and HA2, however, the reactivity of HA2-specific MAbs was remarkably lower as compared with HA1-specific MAbs. Quantitative analysis with two MAbs, IB8 (anti-HA1) and IIF4 (anti-HA2) respectively, revealed that HA2 epitope was reachable for antibody only in minor subpopulation of the HA representing approximately 7% of all molecules (spikes). The basis of the HA heterogeneity is discussed.

Molecular Detection of Murine Herpesvirus 68 in Ticks Feeding on Free-living Reptiles

The MHV-68 (designed as Murid herpesvirus 4 (MuHV 4) strain 68) isolated from two rodents, Myodes glareolus and Apodemus flavicollis, is considered as a natural pathogen of free-living murid rodents. Recently, the detection of MHV antibodies in the blood of animals living in the same biotope as MHV-infected mice has suggested that ticks may have a role in the transmission of this pathogen. Ixodes ricinus is one the most abundant tick species in Europe known to transmit multiple pathogens causing human and animal diseases. In this study, nymphs and larvae feeding on 116 individuals of a temperate lizard species—the green lizard Lacerta viridis captured in the Slovak Karst National Park, were examined for MHV-68. The specific sequence of virion glycoprotein 150 was amplified in DNA individually isolated from I. ricinus ticks using single-copy sensitive nested polymerase chain reaction. MHV-68 was detected in ten of 649 nymphs and in five of 150 larvae, respectively. We found that 9.6% of green lizards fed at least one MHV-68-infected immature tick. Occurrence of MHV-68 within all ticks tested was 1.8%. This study is first to show that immature I. ricinus ticks feeding on free-living lizards in a Central European region could be infected with gammaherpesvirus (MHV-68), naturally infecting free-living murid rodents. Our results provide evidence supporting the hypothesis that ticks may play a mediating role in circulation of MHV-68 in nature.

Reconstitution of carbonic anhydrase activity of the cell-surface-binding protein of vaccinia virus

The N-terminal region of a 32 kDa cell-surface-binding protein, encoded by the D8L gene of vaccinia virus, shows sequence homology to CAs (carbonic anhydrases; EC 4.2.1.1). The active CAs catalyse the reversible hydration of CO2 to bicarbonate participating in many physiological processes. The CA-like domain of vaccinia protein [vaccCA (vaccinia virus CA-like protein)] contains one of the three conserved histidine residues required for co-ordination to the catalytic zinc ion and for enzyme activity. In the present study, we report the engineering of catalytically active vaccCA mutants by introduction of the missing histidine residues into the wild-type protein. The wild-type vaccCA was inactive as a catalyst and does not bind sulfonamide CA inhibitors. Its position on a phylogram with other hCAs (human CAs) shows a relationship with the acatalytic isoforms CA X and XI, suggesting that the corresponding viral gene was acquired from the human genome by horizontal gene transfer. The single mutants (vaccCA N92H/Y69H) showed low enzyme activity and low affinity for acetazolamide, a classical sulfonamide CA inhibitor. The activity of the double mutant, vaccCA N92H/Y69H, was much higher, of the same order of magnitude as that of some human isoforms, namely CA VA and CA XII. Moreover, its affinity for acetazolamide was high, comparable with that of the most efficient human isoenzyme, CA II (in the low nanomolar range). Multiplication of vaccinia virus in HeLa cells transfected with the vaccCA N92H/Y69H double mutant was approx. 2-fold more efficient than in wild-type vaccCA transfectants, suggesting that the reconstitution of the enzyme activity improved the virus life cycle.